Insulin promoter factor, and uses related thereto

ABSTRACT

The present invention relates to the discovery in eukaryotic cells, particularly mammalian cells, of novel a transcriptional regulatory factor, referred to hereinafter as “Insulin Promoter Factor 1” or “Ipf1”.

BACKGROUND OF THE INVENTION

[0001] Each year, over 728,000 new cases of diabetes are diagnosed and150,000 Americans die from the disease and its complications; the totalyearly cost in the United States is over 20 billion dollars (Langer etal. (1993) Science 260:920-926). For instance, diabetes is characterizedby pancreatic islet destruction or dysfunction leading to loss ofglucose control.

[0002] Diabetes mellitus is a metabolic disorder defined by the presenceof chronically elevated levels of blood glucose (hyperglycemia).Insulin-dependent (Type 1) diabetes mellitus (“IDDM”) results from anautoimmune-mediated destruction of the pancreatic β-cells withconsequent loss of insulin production, which results in hyperglycemia.Type 1 diabetics require insulin replacement therapy to ensure survival.Non-insulin-dependent (Type 2) diabetes mellitus (“NIDDM”) is initiallycharacterized by hyperglycemia in the presence of higher-than-normallevels of plasma insulin (hyperinsulinemia). In Type 2 diabetes, tissueprocesses which control carbohydrate metabolism are believed to havedecreased sensitivity to insulin. Progression of the Type 2 diabeticstate is associated with increasing concentrations of blood glucose, andcoupled with a relative decrease in the rate of glucose-induced insulinsecretion.

[0003] The primary aim of treatment in both forms of diabetes mellitusis the same, namely, the reduction of blood glucose levels to as nearnormal as possible. Treatment of Type 1 diabetes involves administrationof replacement doses of insulin. In contrast, treatment of Type 2diabetes frequently does not require administration of insulin. Forexample, initial therapy of Type 2 diabetes may be based on diet andlifestyle changes augmented by therapy with oral hypoglycemic agentssuch as sulfonylurea. Insulin therapy may be required, however,especially in the later stages of the disease, to produce control ofhyperglycemia in an attempt to minimize complications of the disease.

[0004] More recently, tissue-engineering approaches to treatment havefocused on transplanting healthy pancreatic islets, usually encapsulatedin a membrane to avoid immune rejection. Three general approaches havebeen tested in animal models. In the first, a tubular membrane is coiledin a housing that contained islets. The membrane is connected to apolymer graph that in turn connects the device to blood vessels. Bymanipulation of the membrane permeability, so as to allow freediffusionof glucose and insulin back and forth through the membrane, yet blockpassage of antibodies and lymphocytes, normoglycemia was maintained inpancreatectomized animals treated with this device (Sullivan et al.(1991) Science 252:718).

[0005] In a second approach, hollow fibers containing islet cells wereimmobilized in the polysaccharide alginate. When the device was placeintraperitoneally in diabetic animals, blood glucose levels were loweredand good tissue compatibility was observed (Lacey et al. (1991) Science254:1782).

[0006] Finally, islets have been placed in microcapsules composed ofalginate or polyacrylates. In some cases, animals treated with thesemicrocapsules maintained normoglycemia for over two years (Lim et al.(1980) Science 210:908; O'Shea et al. (1984) Biochim. Biochys. Acta.840:133; Sugamori et al. (1989) Trans. Am. Soc. Artif. Intern. Organs35:791; Levesque et al. (1992) Endocrinology 130:644; and Lim et al.(1992) Transplantation 53:1180).

[0007] However, all of these transplantation strategies require a large,reliable source of donor islets.

SUMMARY OF THE INVENTION

[0008] The present invention relates to the discovery in eukaryoticcells, particularly mammalian cells, of novel a transcriptionalregulatory factor, referred to hereinafter as “Insulin Promoter Factor1” or “Ipf1”.

[0009] In general, the invention features an Ipf1 polypeptide,preferably a substantially pure preparation of the polypeptide, or arecombinant Ipf1 polypeptide. In preferred embodiments the polypeptidehas a biological activity associated with its binding to Ipf1-responsiveelements, such as the P1 insulin promoter site, and with its binding toother transcriptional regulatory proteins. The polypeptide can beidentical to the polypeptide shown in SEQ ID No:2, or it can merely behomologous to that sequence. For instance, the polypeptide preferablyhas an amino acid sequence at least 60% homologous to the amino acidsequence in SEQ ID No:2, though higher sequence homologies of, forexample, 80%, 90% or 95% are also contemplated. The polypeptide of thepresent invention can comprise the fall length protein represented inSEQ ID No:2, or it can comprise a fragment of that protein, whichfragment may be, for instance, at least 5, 10, 20, 50 or 100 amino acidsin length. The fragment can be derived to include, for example, regionsof the protein which are likely to be involved in protein-proteininteractions with other transcriptional regulatory proteins or which mayinfluence the DNA-binding specficity of the homeodomain of Ipf1(Glu146-Ser211) relative to other heterologous homeodomains. Forinstance, the fragment can include at least 4 amino acid residuesbetween Met1 to Glu145 and/or Ser212 to Arg284, though more preferablyincludes portions of at least 10, 20, 30 or 50 residues from one or bothof those regions. Exemplary fragments include N-terminal fragmentswithin or including Met1 to Glu145, or C-terminal fragments within orincluding Glu146 to Arg284.

[0010] Moreover, as described below, the Ipf1 polypeptide of the presentinvention can be either an agonist (e.g. mimics), or alternatively, anantagonist of a biological activity of a naturally occurring form ofIpf1. That is, the polypeptide is an Ipf1 homolog which is able tomodulate Ipf1-mediated gene expression (e.g., a gene containing anIpf1-responsive element) in at least one tissue in which wild-type Ipf1is expressed, such as in pancreatic tissue, particularly islet cells.

[0011] In a preferred embodiment, a peptide having at least onebiological activity of the subject polypeptide may differ in amino acidsequence from the sequence in SEQ ID No:2, but such differences resultin a modified protein which functions in the same or similar manner asthe native Ipf1 or which has the same or similar characteristics of thenative Ipf1. Moreover, homologs of the naturally occurring protein arecontemplated which are antagonistic of the normal cellular role of thenaturally occurring form of Ipf1. For example, 1 zips the homolog may becapable of interfering with the ability of wild-type Ipf1 to modulategene expression, e.g. of developmentally or growth regulated genes.Preferred antagonistic forms of an Ipf1 polypeptide either (i) retainsthe DNA binding ability of authentic Ipf1 but lack the ability toassemble transcriptionally-competent protein complexes, or (ii) lacksDNA binding ability (e.g. to Ipf1-RE2) yet retains the ability to bindto other transcription regulatory complexes normally involving authenticIpf1.

[0012] In yet other preferred embodiments, the Ipf1 polypeptide is arecombinant fusion protein which includes a second polypeptide portion,e.g., a second polypeptide having an amino acid sequence unrelated toIpf1, e.g. the second polypeptide portion is glutathione-S-transferase,e.g. the second polypeptide portion is a DNA binding domain of aheterologous transcriptional regulatory protein, or the secondpolypeptide portion is an RNA polymerase activating domain, e.g. thefusion protein is functional in a two-hybrid assay.

[0013] Yet another aspect of the present invention concerns an immunogencomprising an Ipf1 peptide in an immunogenic preparation, the immunogenbeing capable of eliciting an immune response specific for said Ipf1polypeptide. The response can be in the form of a humoral response, e.g.an antibody response or a cellular response. In preferred embodiments,the immunogen comprising an antigenic determinant, e.g. a uniquedeterminant, from a protein represented by SEQ ID No:2.

[0014] A still further aspect of the present invention features anantibody preparation specifically reactive with an epitope of an Ipf1polypeptide, such as an Ipf1 immunogen.

[0015] Another aspect of the present invention provides a substantiallyisolated nucleic acid having a nucleotide sequence which encodes an Ipf1polypeptide. In preferred embodiments: the encoded polypeptidespecifically binds an Ipf1-responsive element, and/or is able to eitheragonize or antagonize assembly of Ipf1-dependent transcriptional proteincomplexes. The coding sequence of the nucleic acid can comprise anIpf1-encoding sequence which can be identical to the cDNA shown in SEQID No:1, or it can merely be homologous to that sequence. For instance,the Ipf1-encoding sequence preferably has a sequence at least 60%homologous to the nucleotide sequence in SEQ ID No:1, though highersequence homologies of, for example, 80%, 90% or 95% are alsocontemplated. The polypeptide encoded by the nucleic acid can comprisethe nucleotide sequence represented in SEQ ID No: 1 which encodes thefull length protein, or it can comprise a fragment of that nucleic acid,which fragment may be, for instance, a fragment of the full length Ipf1protein which is, for example, at least 5, 10, 20, 50 or 100 amino acidsin length. The polypeptide encoded by the nucleic acid can be either anagonist (e.g. mimics), or alternatively, an antagonist of a biologicalactivity of a naturally occurring form of the Ipf1 protein, e.g., thepolypeptide is able to modulate Ipf1-dependent gene expression in atleast one tissue in which the Ipf1 protein is expressed, such as inpancreatic tissue.

[0016] Furthermore, in certain preferred embodiments, the subject Ipf1nucleic acid will include a transcriptional regulatory sequence, e.g. atleast one of a transcriptional promoter or transcriptional enhancersequence, which regulatory sequence is operably linked to the Ipf1genesequence. Such regulatory sequences can be used in to render the Ipf1gene sequence suitable for use as an expression vector.

[0017] In yet a further preferred embodiment, the nucleic acidhybridizes under stringent conditions to a nucleic acid probecorresponding to at least 12 consecutive nucleotides of SEQ ID No:1;preferably to at least 20 consecutive nucleotides of SEQ ID No:1; morepreferably to at least 40 consecutive nucleotides of SEQ ID No:1.

[0018] The invention also features transgenic non-human animals, e.g.mice, rats, rabbits or pigs, having a transgene, e.g., animals whichinclude (and preferably express) a heterologous form of the Ipf1 genesdescribed herein, e.g. a gene derived from humans, or which misexpressan endogenous Ipf1 gene, e.g., an animal in which expression of thesubject Ipf1 protein is disrupted. Such a transgenic animal can serve asan animal model for studying cellular disorders comprising mutated ormis-expressed Ipf1 alleles or for use in drug screening.

[0019] The invention also provides a probe/primer comprising asubstantially purified oligonucleotide, wherein the oligonucleotidecomprises a region of nucleotide sequence which hybridizes understringent conditions to at least 10 consecutive nucleotides of sense orantisense sequence of one of SEQ ID No:1, or naturally occurring mutantsthereof. In preferred embodiments, the probe/primer further includes alabel group attached thereto and able to be detected. The label groupcan be selected, e.g., from a group consisting of radioisotopes,fluorescent compounds, enzymes, and enzyme co-factors. Probes of theinvention can be used as a part of a diagnostic test kit for identifyingβ-islet cells including abnormal β-cells, as well as abnormal pancreatictissues. For instance, the probe can be employed for detecting, in asample of cells isolated from a patient, a level of a nucleic acidencoding the subject Ipf1 protein or mutated forms thereof; e.g.measuring the Ipf1 mRNA level in a cell, or determining whether thegenomic Ipf1 gene has been mutated or deleted. Preferably, theoligonucleotide is at least 10 nucleotides in length, though primers of20, 30, 50, 100, or 150 nucleotides in length are also contemplated.

[0020] In yet another aspect, the invention provides an assay forscreening test compounds for an inhibitor, or alternatively, apotentiator, of an interaction between an Ipf1 and an Ipf1-responsiveelement (such as a P1 promoter), or with other transcriptionalregulatory proteins. An exemplary method includes the steps of (i)combining an Ipf1 protein, a test compound, and an Ipf1-target molecule,under conditions wherein, but for the test compound, the Ipf1protein andthe Ipf1-target molecule are able to interact; and (ii) detecting theformation of a complex which includes the Ipf1 protein and the targetmolecule. A statistically significant change, such as a decrease, in theformation of the complex in the presence of a test compound (relative towhat is seen in the absence of the test compound) is indicative of amodulation, e.g., inhibition, of the interaction between Ipf1 and thetarget molecule. In preferred embodiments, the target molecule is anIpf1-responsive element, e.g., a nucleic acid comprising an Ipf1 bindingsequence, such as an insulin P1 promoter sequence. In alternativeembodiments, the target molecule is a protein which binds Ipf1, such asa protein involved in forming transcriptional regulatory complexes withIpf1. Moreover, primary screens are provided in which the targetmolecule and Ipf1 are combined in a cell-free system and contacted withthe test compound; i.e. the cell-free system is selected from a groupconsisting of a cell lysate and a reconstituted protein:DNA orprotein:protein mixture. Alternatively, the target molecule and Ipf1protein are simultaneously provided in a cell, and the cell is contactedwith the test compound. For example, where the target molecule is anucleic acid comprising an Ipf1-responsive element, the expression of amarker gene controlled by the Ipf1-responsive element is detected.

[0021] The present invention also provides a method for treating ananimal, including a human, having a disorder characterized by a loss of,or abnormal control of, wild-type function of Ipf1, comprisingadministering an effective amount of an Ipf1 agonist. In one embodiment,the method comprises administering a nucleic acid construct encoding apolypeptide represented in SEQ ID No:2, under conditions wherein theconstruct is incorporated by cells deficient in insulin production, andunder conditions wherein the recombinant gene is expressed, e.g. by genetherapy techniques. In other embodiments, the action of anaturally-occurring Ipf1 protein is antagonized by therapeuticexpression of an Ipf1 homolog which is an antagonistic of, for example,assembly of functional Ipf1 transcriptional regulatory complexes, or bydelivery of an antisense nucleic acid molecule which inhibits IPF1transcriptional regulation. Such techniques can likewise be used totreat a disorder characterized by abherent or unwanted expression of agene regulated by an Ipf1-RE, such as an insulin gene.

[0022] Another aspect of the present invention provides a method ofdetermining if a subject, e.g. a human patient, is at risk for adisorder characterized by unwanted cell proliferation ordifferentiation. The method includes detecting, in a tissue of thesubject, the presence or absence of a genetic lesion characterized by atleast one of (i) a mutation of a gene encoding a protein represented bySEQ ID No:2, or a homolog thereof; (ii) the mis-expression of a geneencoding a protein represented by SEQ ID No:2; or (iii) themis-incorporation of Ipf1in a transcriptional regulatory complex. Inpreferred embodiments: detecting the genetic lesion includesascertaining the existence of at least one of: a deletion of one or morenucleotides from the Ipf1 gene; an addition of one or more nucleotidesto the gene, an substitution of one or more nucleotides of the gene, agross chromosomal rearrangement of the gene; an alteration in the levelof a messenger RNA transcript of the gene; the presence of a non-wildtype splicing pattern of a messenger RNA transcript of the gene; or anon-wild type level of the protein.

[0023] For example, detecting the genetic lesion can include (i)providing a probe/primer including an oligonucleotide containing aregion of nucleotide sequence which hybridizes to a sense or antisensesequence of SEQ ID No:1, or naturally occurring mutants thereof or 5′ or3′ flanking sequences naturally associated with the Ipf1 gene; (ii)exposing the probe/primer to nucleic acid of the tissue; and (iii)detecting, by hybridization of the probe/primer to the nucleic acid, thepresence or absence of the genetic lesion; e.g. wherein detecting thelesion comprises utilizing the probe/primer to determine the nucleotidesequence of the Ipf1 gene and, optionally, of the flanking nucleic acidsequences. For instance, the probe/primer can be employed in apolymerase chain reaction (PCR) or in a ligation chain reaction (LCR).In alternate embodiments, the level of Ipf1 protein and/or itsparticipation in complexes is detected in an immunoassay using anantibody which is specifically immunoreactive with a protein representedby SEQ ID No:2.

[0024] The invention also features transgenic non-human animals, e.g.mice, rats, rabbits or pigs, harboring in one or more of its cells anIpf1-encoding transgene. In preferred embodiments, the transgene isexpressed, causing Ipf1-dependent gene transcription where the transgeneencodes an agonistic form of the protein, or disruption of Ipf1-inducedexpression where the transgene encodes an antagonistic form of theprotein. Such transgenic animals can serve as models for studyingcellular and tissue disorders comprising mutated or mis-expressed Ipf1alleles, as well as for studying the physiological role of Ipf1 inproliferation, differentiation and maintenance of tissues in vivo inboth adult and embryonic systems. Furthermore, inhibition of Ipf1expression in certain cells, such as β-cells, can be used to unravel theeffects of various autocrine and paracrine functions of pancreatichormones, and can be used in drug screening assays designed to detectmodulators of these other factors.

[0025] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.3. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).Other references include Ohlsson et al. (1993) EMBO J 12:4251-4259;Ohlsson et al. (1991) Mol Endo 5:897-904; Walker et al. (1983) Nature306:557-561; Leonard et al. (1993) Mol Endo 7:1275-1283; Miller et al.(1994) EMBO J 13:1145-1156; and Harrison et al. (1994) J Biol Chem269:19968-19975, all of which are incorporated by reference herein.

[0026] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

[0027]FIGS. 1A, 1B and 1C illustrate the results of expression of theIpf1 gene in both β-cells and non-β-cells transactivates a reporterconstruct via the P1 site.

[0028] (FIG. 1A) Mutation of the P1 promoter site results in a decreasedactivity for the rat insulin I 5′ flank and the stimulation of theactivity of the 5′ flank as a result of Ipf1 expression is criticallydependent on an intact P1 site. RSV and RSV/Ipf1 recombinant expressionvectors were co-transfected with the Tk-CAT, Ins-CAT and P1mut#2/Ins-CAT reporter genes into βTC1 cells using an internal controlβ-gal plasmid as described previously (Walker et al. (1983) Nature306:557-581).

[0029] (FIG. 1B) Multimers of the P1 site in front of a reporter gene isspecifically trans-activated by the expression of the Ipf1 gene inheterologous cells. RSV, RSV-Ipf1 and RSV-Isl1 recombinant expressionvectors were co-transfected with the β-globin-CAT and 5× P1 β-globin-CATconstruct in CHO cells.

[0030] (FIG. 1C) Overexpression of the Ipf1 gene in PTC1 cells resultsin a further up-regulation of the activity of the P1 element. RSV andRSV-Ipf1recombinant expression vectors were co-transfected with theβ-globin-CAT and 5× P1 β-globin-CAT construct in βTC1 cells. The numbersgiven are normalized to the internal control and represent the mean ofat least five independent transfection experiments. In all cases, thestandard error of the mean was <1 5% of this value.

[0031]FIG. 2 is a schematic representation of targeting construct,genomic DNA and the expected product of homologous recombination. Thetwo exons of Ipf1 are indicated by cylinders and the bacterial neomycingene, under control of the herpes simplex virus (HSV) promoter/enhancer,is represented by a triangular bar. Deletion of the 3.1-kb HindIII/NcoIfragment from within the 7.2-kb BamHI segment of the Ipf1 genomic DNAresults in loss of the entire homeobox, the splice acceptor site andparts of the intron. This fragment was replaced with the 1,142-bpXnoI/BamHI fragment from pMC1 neopoly(A) (Thomas et al. (1987) Cell51:503-512). Restriction enzymes: B, BamHI; H, HindIII; N. NcoI; P.PstI. The mouse Ipf1 gene was cloned from a mouse 129/SV genomiclibrary. E14-1 ES cells were cultured on mitomycin-treated embryonicfibroblasts in medium supplemented with 1,000 U/ml leukaemia-inhibitoryfactor as previously described (Kuhn et al. (1991) Science 254:707-710).A Bio-Rad GenePulser at 500 F, 260 mV was used to electroporate 10⁷cells with 25 μg ml linearized targeting DNA. Cells were plated onmitomycin-treated neomycin-resistant STO fibroblasts. Selection with 250μg ml G418 was initiated after 48 h and ES colonies were picked eightdays later. Blastocysts from C57BL/6 mice were injected and transferredto pseudopregnant (C57BL/6× CBA)F₁ females to generate chimaericoffspring as described in Hogan et al. in Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

DETAILED DESCRIPTION OF THE INVENTION

[0032] The endocrine pancreas of mammals is composed of several thousandislets of Langherhans. Each individual islet contains fourhormone-producing cell types in a characteristic proportion anddistribution, with the different hormone-producing cells appearingsequentially during embryo genesis (Pictet et al. (1972) in Steiner, D.F. and Frenkel, M. (EDS), Handbook of Physiology, Series 7, AmericanPhysiology Society, Washington, D.C., pp. 25-66; Yoshinari et al. (1992)Anat. Embryol. 165:63-70; Titelman et al. (1987) Dev. Biol. 121:454-466;Herrera et al. (1991) Development 113:1257-1265; Gitts et al. (1992)PNAS 89:1128-1132). Although the precise lineage relationship betweenthe different islet cells is not known, co-expression of differenthormone genes during normal pancreas development and in clonedcell-lines derived from islet cell tumors suggests a common precursorfor the pancreatic endocrine cells (Medsen et al. (1986) J. Cell Biol.103:2025-2034; Alpert et al. (1988) Cell 53:295-308; and Herrera et al.Supra). These observations have suggested that terminal differentiation,restricting the expression of the hormone genes to the individualendocrine cell-type, occurs relatively late in the ontogeny of theendocrine pancreas.

[0033] For some of these hormone genes it has been possible to identifythe cis- and trans-acting elements that regulate the islet-specificexpression of the genes. For instance, the insulin-1 gene containsapproximately 350 basepairs of 5′ flanking DNA (e.g., the “insulintranscriptional regulatory sequence”) which is sufficient for selective,β-cell specific expression both in cell lines and in transgenic animals(Walker et al. (1983) Nature 306:557-581; and Alpert et al., supra),with both a strong β-cell enhancer and a promoter element containedwithin these 350 basepairs (Edlund et al. (1983) Science 230:912-916;and Karlson 203 et al. (1987) PNAS 84:8819-8823).

[0034] This invention, as described below, derives in part from thecloning of a mamalian transcriptional regulatory protein which binds toand activates transcription from the insulin transcriptional regulatorysequence. This transcriptional regulatory factor, referred tohereinafter as “Insulin Promoter Factor 1” or “Ipf1” is apparently partof the mechanism involved in developmental coordination of endodermdifferentiation, particularly of the pancreas and other tissues derivedfrom the primative gut. For instance, as described in the appendedexamples, analysis of Ipf1 expression patterns demonstrate that Ipf1expression occurs in the developing foregut endoderm when this tissuecommits to a pancreatic fate.

[0035] Moreover, transgenic animals in which Ipf1 expression isdisrupted selectively lack a pancreas. These findings show that Ipf1 isneeded for the formation of the pancreas, and strongly implicates Ipf1function in the determination and/or maintenance of the pancreaticidentity of common precursor cells, and/or in the regulation of theirpropagation. Ipf1-mediated gene expression is presumably important inthe pathogenesis of diabetes and other abnormal glycemic disease states,and may also be of significance in the progression and pathology ofother proliferative or differentiative disorders. Consequently, theinteraction of Ipf1 with Ipf1-responsive elements, as well as with otherregulatory proteins, may be significant in the modulation of cellularhomeostasis, in the control of organogenesis, and/or in the maintenanceof differentiated tissues, as well as in the development of tissuefailure and neoplastic disorders.

[0036] Accordingly, certain aspects of the present invention relate todiagnostic and therapeutic assays and reagents for detecting andtreating disorders involving abherent assembly of Ipf1 transcriptionalcomplexes. Moreover, drug discovery assays are provided for identifyingagents which can modulate the binding of Ipf1 with other transcriptionalregulatory proteins or with Ipf1 responsive elements. Such agents can beuseful therapeutically to alter the growth and/or differentiation ofpancreatic cell. Other aspects of the invention are described below orwill be apparent to those skilled in the art in light of the presentdisclosure.

[0037] For convenience, certain terms employed in the specification,examples, and appended claims are collected here.

[0038] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single-stranded(such as sense or antisense) and double-stranded polynucleotides.

[0039] As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding an insulinpromoter factor of the present invention, including both exon and(optionally) intron sequences. A “recombinant gene” refers to nucleicacid encoding Ipf1 and comprising Ipf1-encoding exon sequences, thoughit may optionally include intron sequences which are either derived froma chromosomal Ipf1 gene or from an unrelated chromosomal gene. Anexemplary Ipf1 recombinant gene is represented by any one of SEQ IDNo:1. The term “intron” refers to a DNA sequence present in a given Ipf1gene which is not translated into protein and is generally found betweenexons.

[0040] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of Ipf1, or whereanti-sense expression occurs, from the transferred gene, the expressionof a naturally-occurring form of Ipf1 is disrupted.

[0041] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of preferred vector is an episome, i.e., a nucleicacid capable of extra-chromosomal replication. Preferred vectors arethose capable of autonomous replication and/expression of nucleic acidsto which they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome. In the present specification, “plasmid”and “vector” are used interchangeably as the plasmid, is the mostcommonly used form of vector. However, the invention is intended toinclude such other forms of expression vectors which serve equivalentfunctions and which become known in the art subsequently hereto.

[0042] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. In preferred embodiments, transcription of a recombinant Ipf1gene is under the control of a promoter sequence (or othertranscriptional regulatory sequence) which controls the expression ofthe recombinant gene in a cell-type in which expression is intended. Itwill also be understood that the recombinant gene can be under thecontrol of transcriptional regulatory sequences which are the same orwhich are different from those sequences which control transcription ofa naturally-occurring form of Ipf1.

[0043] As used herein, the term “Ipf1-responsive element” or “Ipf1-RE”refers to a 2G transcriptional regulatory sequence which controlsexpression of a gene in an Ipf1-dependent manner. That is, the Ipf1-REhas a nucleotide sequence which is specifically bound by an Ipf1protein, and the binding of Ipf1 regulates expression of a gene operablylinked to the Ipf1-RE. An exemplary Ipf1-RE is the 5′ flankingtranscriptional regulation DNA of the insulin I gene, particularly thePI promoter site 5′-GCCCTTAATGGGCCAA, or its core sequence TAATGGG.

[0044] As used herein, the term “tissue-specific promoter” means a DNAsequence that serves as a promoter, i.e., regulates expression of aselected DNA sequence operably linked to the promoter, and which effectsexpression of the selected DNA sequence in specific cells of a tissue,such as cells of a urogenital origin. e.g. renal cells, or cells of aneural origin. e.g. neuronal cells. The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well.

[0045] As used herein, a “transgenic animal” is any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by trangenic techniques well known in theart. The nucleic acid is introduced into the cell, directly or sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

[0046] “Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0047] A “chimeric protein” or “fusion protein” is a fusion of a firstamino acid sequence encoding an Ipf1 polypeptide with a second aminoacid sequence defining a domain foreign to and not substantiallyhomologous with any domain of the subject Ipf1. A chimeric protein maypresent a foreign domain which is found (albeit in a different protein)in an organism which also expresses the first protein, or it may be an“interspecies”, “intergenic”, etc. fusion of protein structuresexpressed by different kinds of organisms.

[0048] The term “evolutionarily related to”, with respect to nucleicacid sequences encoding Ipf1, refers to nucleic acid sequences whichhave arisen naturally in an organism, including naturally occurringmutants. The term also refers to nucleic acid sequences which, whilederived from a naturally occurring Ipf1, have been altered bymutagenesis, as for example, combinatorial mutagenesis described below,yet still encode polypeptides which have at least one activity of aIpf1.

[0049] The term “isolated” as also used herein with respect to nucleicacids, such as DNA or RNA, refers to molecules separated from otherDNAs. or RNAs, respectively, that are present in the natural source ofthe macromolecule. For example, an isolated nucleic acid encoding one ofthe subject Ipf1 preferably includes no more than 10 kilobases (kb) ofnucleic acid sequence which naturally immediately flanks that particularIpf1 gene in genomic DNA, more preferably no more than 5 kb of suchnaturally occurring flanking sequences, and most preferably less than1.5 kb of such naturally occurring flanking sequence. The term isolatedas used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state.

[0050] As described below, one aspect of the invention pertains to anisolated nucleic acid comprising the nucleotide sequence encoding anIpf1 polypeptide, and/or equivalents of such nucleic acids. The termnucleic acid as used herein is intended to include fragments andequivalents. The term equivalent is understood to include nucleotidesequences encoding functionally equivalent Ipf1 molecules orfunctionally equivalent polypeptides which, for example, retain theability to bind to other transcriptional regulatory proteins or totranscriptional regulatory sequences of, for example, an insulin gene.Equivalent nucleotide sequences will include sequences that differ byone or more nucleotide substitutions, additions or deletions, such asallelic variants; and will, therefore, include sequences that differfrom the nucleotide sequence of Ipf1 shown in SEQ ID No:1 due to thedegeneracy of the genetic code. Equivalents will also include nucleotidesequences which hybridize under stringent conditions (i.e., equivalentto about 20-27° C. below the melting temperature (T_(m)) of the DNAduplex formed in about 1M salt) to the nucleotide sequence representedin SEQ ID No:1. In one embodiment, equivalents will further includenucleic acid sequences derived from or otherwise related to, thenucleotide sequence shown SEQ ID No:1.

[0051] For example, it will be generally appreciated that, under certaincircumstances, it may I be advantageous to provide homologs of thesubject Ipf1 protein which, while not identical to SEQ ID No.:2,function as an Ipf1 agonist or an Ipf1 antagonist, in order to promoteor inhibit the biological activities of the naturally-occurring form ofthe protein. For instance, antagonistic homologs can be generated whichinterfere with the ability of wild-type (“authentic”) Ipf1 to formtranscriptional activating complexes at Ipf1-responsive elements. Asdescribed below, an antagonistic Ipf1 homolog, such as a truncationmutant which retains DNA-binding activity yet is transcriptionallydefective, can be used in the treatment of, for example,hyperinsulinemia A polypeptide is considered to possess a biologicalactivity of Ipf1 if the polypeptide has one or more of the followingproperties: the ability to modulate at least one of proliferation,differentiation or survival of a cell which expresses a gene that istranscriptionally regulated by an Ipf1-RE; the ability to modulate geneexpression of a gene that is transcriptionally regulated by an Ipf1-RE,e.g. of a developmentally or growth regulated gene, e.g. of an insulingene; the ability to modulate gene expression in pancreatic tissue, e.g.in the ability to bind to the ability agonize or antagonize assembly ofIpf1-containing transcriptional protein complexes. An Ipf1 polypeptidemay additionally be characterized by the ability to modulatedifferentiation of endodermally-derived tissue, such as tissue derivedfrom the primitive gut, e.g. pancreatic tissue, e.g. β-cells. A proteinalso has Ipf1 biological activity if it is a specific agonist orantagonist of one of the above recited properties.

[0052] Preferred nucleic acids encode an Ipf1 polypeptide comprising anamino acid sequence at least 60% homologous, more preferably 70%homologous and most preferably 80% homologous with an amino acidsequence shown in one of SEQ ID No:2. Nucleic acids which encodepolypeptides that retain an activity of Ipf1 and having at least about90%, more preferably at least about 95%, and most preferably at leastabout 98-99% homology with a sequence shown in one of SEQ ID No:2 arealso within the scope of the invention, as of course are proteins whichare identical to the aforementioned sequence listings. In oneembodiment, the nucleic acid is a cDNA encoding a peptide having atleast one activity of the subject Ipf1 protein. Preferably, the nucleicacid is a cDNA molecule comprising at least a portion of the nucleotidesequence represented in one of SEQ ID No:1. A preferred portion of thesecDNA molecules includes the coding region of the gene.

[0053] Another aspect of the invention provides a nucleic acid whichhybridizes under high or low stringency conditions to a DNA or RNA whichencodes a peptide having all or a portion of the amino acid sequenceshown in SEQ ID No:2. Appropriate stringency conditions which promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0× SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0× SSC at 50° C. to a high stringency of about0.2× SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C.

[0054] Nucleic acids which have a sequence that differ from thenucleotide sequence shown in SEQ ID No:1 due to degeneracy in thegenetic code are also within the scope of the invention. Such nucleicacids encode functionally equivalent peptides (i.e., a peptide having abiological activity of a Ipf1) but that differ in sequence from saidsequence listings due to degeneracy in the genetic code. For example, anumber of amino acids are designated by more than one triplet. Codonsthat specify the same amino acid, or synonyms (for example, CAU and CACeach encode histidine) may result in “silent” mutations which do notaffect the amino acid sequence of Ipf1 polypeptide. However, it isexpected that DNA sequence polymorphisms that do lead to changes in theamino acid sequences of the subject Ipf1 will exist among vertebrates.One skilled in the art will appreciate that these variations in one ormore nucleotides (up to about 3-5% of the nucleotides) of the nucleicacids encoding Ipf1polypeptides Ipf1 may exist among individuals of agiven species due to natural allelic variation. Any and all suchnucleotide variations and resulting amino acid polymorphisms are withinthe scope of this invention.

[0055] Fragments of the nucleic acid encoding the subject Ipf1 are alsowithin the scope of the invention. As used herein, a fragment encodingthe active portion of a Ipf1 refers to a nucleic acid having fewernucleotides than the nucleotide sequence encoding the entire amino acidsequence of Ipf1 but which nevertheless encodes a peptide which iseither an agonist or antagonist of authentic Ipf1, e.g. the fragmentretains the ability to bind to an insulin promoter. Nucleic acidfragments within the scope of the present invention include thosecapable of hybridizing under high or low stringency conditions withnucleic acids from other species for use in screening protocols todetect Ipf1 homologs, including alternate isoforms, e.g. mRNA splicingvariants. Nucleic acids within the scope of the invention may alsocontain linker sequences, modified restriction endonuclease sites andother sequences useful for molecular cloning, expression or purificationof recombinant forms of the subject Ipf1protein.

[0056] As indicated by the examples set out below, a nucleic acidencoding Ipf1 or a homologous gene thereof may be obtained from MRNApresent in any of a number of eukaryotic cells. It should also bepossible to obtain nucleic acids encoding Ipf1 from genomic DNA obtainedfrom both adults and embryos. For example, a gene encoding Ipf1can becloned from either a cDNA or a genomic library in accordance withprotocols herein described, as well as those generally known to personsskilled in the art. A cDNA encoding a Ipf1 can be obtained by isolatingtotal mRNA from a cell, e.g. a mammalian cell, e.g. a human cell. Doublestranded cDNAs can then be prepared from the total mRNA, andsubsequently inserted into a suitable plasmid or bacteriophage vectorusing any one of a number of known techniques. The gene encoding theIpf1 can also be cloned using established polymerase chain reactiontechniques in accordance with the nucleotide sequence informationprovided by the invention. The nucleic acid of the invention can be DNAor RNA. A preferred nucleic acid is a cDNA represented by the sequenceshown in SEQ ID No:1.

[0057] Another aspect of the invention relates to the use of theisolated nucleic acid in “antisense” therapy. As used herein,“antisense” therapy refers to administration or in situ generation ofoligonucleotide probes or their derivatives which specificallyhybridizes (e.g. binds) under cellular conditions, with the cellularmRNA and/or genomic DNA encoding an Ipf1 protein so as to inhibitexpression of that protein, e.g. by inhibiting transcription and/ortranslation. The binding may be by conventional base paircomplementarity, or. for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

[0058] An antisense construct of the present invention can be delivered,for example, as an expression plasmid which, when transcribed in thecell, produces RNA which is complementary to at least a unique portionof the cellular mRNA which encodes an Ipf1 protein. Alternatively, theantisense construct can be an oligonucleotide probe which is generatedex vivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of anIpf1 gene. Such oligonucleotide probes are preferably modifiedoligonucleotides which are resistant to endogenous nucleases, e.g.exonucleases and/or endonucleases, and is therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example, by van der Krol etal. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668.

[0059] Accordingly, the modified oligomers of the invention are usefulin therapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general. For such therapy, the oligomers of theinvention can be formulated for a variety of loads of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in id Remmington'sPharmaceutical Sciences, Meade Publishing Co., Easton, Pa., and mayinclude both human and vetinary formulations. For systemicadministration, injection is preferred, including intramuscular,intravenous, intraperitoneal, and subcutaneous for injection, theoligomers of the invention can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the oligomers may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

[0060] In addition to use in therapy, the oligomers of the invention maybe used as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are described in further detail below.

[0061] Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of Ipf1 (by inhibiting itsexpression), can be used in the manipulation of tissue, e.g. tissuedifferentiation, both in vivo and in ex vivo tissue cultures, as well asin the treatment of hyperinsulinenemia, such as during various stages ofnon-insulin dependent 2) diabetes mellitus.

[0062] This invention also provides expression vectors containing anucleic acid encoding an Ipf1 polypeptide, operably linked to at leastone transcriptional regulatory sequence. Operably linked is intended tomean that the nucleotide sequence is linked to a regulatory sequence ina manner which allows expression of the nucleotide sequence. Regulatorysequences are art-recognized and are selected to direct expression of arecombinant Ipf1polypeptide. Accordingly, the term transcriptionalregulatory sequence includes promoters, enhancers and other expressioncontrol elements. Such regulatory sequences are described in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). For instance, any of a wide variety ofexpression control sequences-sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding the Ipf1 proteins of this invention.Such useful expression control sequences, include, for example, theearly and late promoters of SV40, adenovirus or cytomegalovirusimmediate early promoter, the lac system, the trp system, the TAC or TRCsystem, T7 promoter whose expression is directed by T7 RNA polymerase,the major operator and promoter regions of phage lambda, the controlregions for fd coat protein, the promoter for 3-phosphoglycerate kinaseor other glycolytic enzymes, the promoters of acid phosphatase, e.g.,Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the Vs type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinsencoded by the vector, such as antibiotic markers, should also beconsidered. In one embodiment, the expression vector includes arecombinant gene encoding a polypeptide which mimics or otherwiseagonizes the action of Ipf1, or alternatively, which encodes apolypeptide that antagonizes the action of an authentic Ipf1. Suchexpression vectors can be used to transfect cells and thereby produceand (optionally) purify proteins, including fusion proteins or peptides,encoded by nucleic acids as described herein.

[0063] Moreover, the gene constructs of the present invention can alsobe used as a part of a gene therapy protocol to deliver nucleic acidsencoding either an agonistic or antagonistic form of the subject Ipf1proteins. Thus, another aspect of the invention features expressionvectors for in vivo transfection and expression of an Ipf1 polypeptidein particular cell types so as to reconstitute the function of, oralternatively, abrogate the function of Ipf1 in a cell in which thatprotein or other transcriptional regulatory proteins to which it bindsare misexpressed. For example, gene therapy can be used to deliver agene encoding an Ipf1protein which promotes insulin expression, such asin the generation of β-cells.

[0064] Expression constructs of the subject Ipf1 proteins, and mutantsthereof, may be administered in any biologically effective carrier, e.g.any formulation or composition capable of effectively delivering theIpf1 gene to cells in vivo. Approaches include insertion of the subjectgene in viral vectors including recombinant retroviruses, adenovirus,adeno-associated virus, and herpes simplex virus-l or recombinantbacterial or eukaryotic plasmids. Viral vectors transfect cellsdirectly; plasmid DNA can be delivered with the help of, for example,cationic liposomes (lipofectin) or derivatized (e.g. antibodyconjugated), polylysine conjugates, gramacidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo. It will be appreciated that because transduction of appropriatetarget cells represents the critical first step in gene therapy, choiceof the particular gene delivery system will depend on such factors asthe phenotype of the intended target and the route of administration,e.g. locally or systemically. Furthermore, it will be recognized thatthe particular gene construct provided for in vivo transduction of Ipf1expression are also useful for in vitro transduction of cells, such asfor use in a diagnostic assays.

[0065] A preferred approach for in vivo introduction of nucleic acidinto a cell is by use of a viral vector containing nucleic acid, e.g. acDNA, encoding the Ipf1 polypeptide or homolog thereof. Infection ofcells with a viral vector has the advantage that a large proportion ofthe targeted cells can receive the nucleic acid. Additionally, moleculesencoded within the viral vector, e.g., by a cDNA contained in the viralvector, are expressed efficiently in cells which have taken up thevector.

[0066] Retrovirus vectors and adeno-associated virus vectors aregenerally understood to be the recombinant gene delivery system ofchoice for the transfer of exogenous genes in vivo, particularly intohumans. These vectors provide efficient delivery of genes into cells,and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host. A major prerequisite for the use ofretroviruses is to ensure the safety of their use, particularly A) withregard to the possibility of the spread of wild-type virus in the cellpopulation. The development of specialized cell lines (termed “packagingcells”) which produce only replication-defective retroviruses hasincreased the utility of retroviruses for gene therapy. and defectiveretroviruses are well characterized for use in gene transfer for genetherapy purposes (for a review see Miller, A. D. (1990) Blood 76:271).Thus, recombinant retrovirus can be constructed in which part of theretroviral coding sequence (gag, pol, env) has been replaced by nucleicacid encoding an Ipf1 polypeptide, rendering the retrovirus replicationdefective. The replication defective retrovirus is then packaged intovirions which can be used to infect a target cell through the use of ahelper virus by standard techniques. Protocols for producing recombinantretroviruses and for infecting cells in vitro or in vivo with suchviruses can be found in Current Protocols in Molecular Biology, Ausubel,F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections9.10-9.14 and other standard laboratory manuals. Examples of suitableretroviruses include pLJ, pZIP, pWE and pEM which are well known tothose skilled in the art. Examples of suitable packaging virus lines forpreparing both ecotropic and amphotropic retroviral systems includeψCrip, ψCre, ψ2 and ψAm. Retroviruses have been used to introduce avariety of genes into many different cell types. including epithelialcells, in vitro and/or in vivo (see for example Eglitis, et al. (1985)Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci.USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1 992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

[0067] Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J. BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety receptor-ligand drug, aswell as by generating fusion proteins (e.g. single-chain antibody/envfusion proteins). For example, agents which bind to β-cell receptors(either ligand or antibody) can be used to enhance infection of β-cells.To illustrate, derivatization of the viral particle with ligands for atleast one of the gluca gon-like peptide receptor (GLP), the sulfonylureareceptor, the galanin receptor,. or antibodies against β-cell antigens,such as GAD65. This technique, while useful to limit or otherwise directthe infection to pancreatic tissue, can also be used to convert anecotropic vector in to an amphotropic vector.

[0068] Another viral gene delivery system useful in the presentinvention utilitizes adenovirus-derived vectors. The genome of anadenovirus can be manipulated such that it encodes and expresses a geneproduct of interest but is inactivated in terms of its ability toreplicate in a normal lytic viral life cycle. See for example Berkner etal. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 d1324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known tothose skilled in the art. The virus particle is relatively stable andamenable to purification and concentration, and as above, can bemodified so as to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use and thereforefavored by the present invention are deleted for all or parts of theviral E1 and E3 genes but retain as much as 80% of the adenoviralgenetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner etal., supra; and Graham et al. in Methods in Molecular Biology, E. J.Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127).Expression of the inserted Ipf1 gene can be under control of, forexample, the EIA promoter, the major late promoter (MLP) and associatedleader sequences, the E3 promoter, or exogenously added promotersequences.

[0069] Yet another viral vector system useful for delivery of thesubject Ipf1 genes is the adeno-associated virus (AAV). Adeno-associatedvirus is a naturally occurring defective virus that requires anothervirus, such as an adenovirus or a herpes virus, as a helper virus forefficient replication and a productive life cycle. (For a review seeMuzyczka et al. Curr. Topics in Micro. and Immunol. (1992) 158:97-129).It is also one of the few viruses that may integrate its DNA intonon-dividing cells, and exhibits a high frequency of stable integration(see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol.7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlinet al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as300 base pairs of AAV can be packaged and can integrate. Space forexogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can beused to introduce Ipf1 genes into cells. A variety of nucleic acids havebeen introduced into different cell types using AAV vectors (see forexample Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

[0070] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed to cause expression of anIpf1 polypeptide in the tissue of an animal. Most nonviral methods ofgene transfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments. non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the subject Ipf1 gene bythe targeted cell. Exemplary gene delivery systems of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes.

[0071] In a representative embodiment, a therapeutic Ipf1 gene can beentrapped in liposomes bearing positive charges on their surface (e.g.,lipofectins) and (optionally) which are tagged with antibodies orligands for pancreatic cell surface antigens (Mizuno et al (1992) NoShinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patentapplication 1047381; and European patent publication EP-A-43075). Forexample, lipofection of β-cells can be carried out using liposomestagged with monoclonal antibodies against, for example, the GAD65antigen, or any other cell surface antigen present on these pancreaticcells. Alternatively, liposomes can be derivative with such receptorligands glimepiride, glibenclamide or other sulfonylurea drug.

[0072] In clinical settings, the gene delivery systems for therapeuticIpf1 genes can be introduced into a patient (or non-human animal) by anyof a number of methods, each of which is familiar in the art. Forinstance, a pharmaceutical preparation of the gene delivery system canbe introduced systemically, e.g. by intravenous injection, and specifictransduction of the protein in the target cells occurs predominantlyfrom specificity of transfection provided by the gene delivery vehicle,cell-type or tissue-type expression due to the transcriptionalregulatory sequences controlling expression of the receptor gene, or acombination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle can beintroduced into the pancreas by catheter (see U.S. Pat. No. 5,328,470),by stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3054-3057),or by electroporation during a partial pancreatectomy (Dev et al.((1994) Cancer Treat Rev 20:105-115).

[0073] The pharmaceutical preparation of the gene therapy construct canconsist essentially of the gene delivery system in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery system can be produced in tact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can comprise one ormore cells which produce the gene delivery system.

[0074] There are a wide variety of pathological cell proliferative anddifferentiative conditions for which the Ipf1 gene constructs of thepresent invention may provide therapeutic benefits, with the generalstrategy being, for example, the correction of abherent insulinexpression, or modulation of differentiative events mediated by Ipf1,such as may be influenced by transcriptional regulatory sequences ofother genes with which the subject Ipf1 interact. More generally.however, the present invention relates to a method of inducing and/ormaintaining a differentiated state, enhancing survival and/or affectingproliferation of a cell in which Ipf1 responsive genes are expressed, bycontacting the cell with an agent which modulates the function (as anagonist or an antagonist) of Ipf1. For instance, it is contemplated bythe invention that, in light of the apparent involvement of Ipf1 in theformation of ordered spatial arrangements of pancreatic tissues, thesubject method could be used to generate and/or maintain such tissueboth in vitro and in vivo. For instance, modulation of the function ofIpf1 can be employed in both cell culture and therapeutic methodsinvolving generation and maintenance β-cells and possibly also fornon-pancreatic tissue, such as in controlling the development andmaintenance of tissue from the digestive tract, spleen, lungs, and otherorgans which derive from the primitive gut. The agent can be, asappropriate, any of the preparations described herein, including genetherapy constructs, antisense molecules, peptidomimetics or other agentsidentified in the drug screening assays provided herein.

[0075] In an exemplary embodiment, the present method can be used in thetreatment of hyperplastic and neoplastic disorders effecting pancreatictissue, particularly those characterized by abherent proliferation ofβ-cells, or mis-expression of Ipf1 or other proteins involved inregulatory complexes involving Ipf1. For instance, pancreatic cancersare marked by abnormal proliferation of pancreatic cells which canresult in alterations of insulin secretory capacity of the pancreas. Forinstance, certain pancreatic hyperplasias, such as pancreaticcarcinomas, can result in hypoinsulinemia due to dysfunction of β-cellsor decreased islet cell mass. Stimulation of Ipf1-mediated expression ofinsulin, such as by overexpression of exogenous Ipf1 in β-cells, can beused to increase the insulin production of normal β-cells in the tissue,as well as enhance regeneration of the tissue after anti-tumor therapy.

[0076] In contrast, other pancreatic tumors, such as islet tumors (e.g.,insulinomas), are marked by overproduction of insulin (i.e.,hyperinsulinemia), which can cause hypoglycemic conditions in a patient.Indeed, hypoglycemia can result from any one of a number of differentdisorders which result in raised plasma insulin levels, including otherβ-cell abnormalities, as well as endocrinopathies, sepsis (includingmalaria), congestive cardiac failure, hepatic and renal insufficiencies,various genetic abnormalities of metabolism, and exogenous toxins (suchas alcohol). According to the present invention, hypoglycemic conditionscan be treated by administering therapeutic amounts of an agent able toantagonize Ipf1-mediated expression of insulin. Depending on the desiredhalf-life of the effects of the treatment, such agents can range frompeptidomimetic and other small molecule inhibitors of Ipf1 function, toantisense constructs, to transient or long-term gene therapy regimens.

[0077] Furthermore, the subject method can be used as part of treatmentsfor various forms of diabetes, as well as other pathologies resultingfrom direct physical/chemical damage to β-cells which result in necrosisand loss of functional islet tissue. In diabetes mellitus, insulinsecretion is either completely absent (IDDM) or inappropriatelyregulated (NIDDM). However, each is characterized by the presence ofchronically elevated levels of blood glucose (hyperglycemia). Theprimary aim of treatment in both forms is the same, namely, thereduction of blood glucose levels to as near as normal as possible. Forexample, treatment of IDDM typically involves administration ofreplacement doses of insulin. In constrast, initial therapy for NIDDMmay be based in part on therapies which include administration ofhypoglycemic agents such as sulfonylurea, though insulin treatment inlater stages of the disease may be required to effect normoglycemia.Accordingly, the present method can provide a means for controllingdiabetogenous glycemic levels, by administeration of an Ipf1 agonist(e.g. a hyperglycemic agent) as, for example, by causing recombinantexpression of a wild-type form of the protein in β-islet cells of thepatient, or alternatively, admininstration of an Ipf1 antagonist (e.g. ahypoglycemic agent) such q a molecule which inhibits response elementbinding and/or activation of insulin gene transcription by Ipf1 orIpf1-containing complexes.

[0078] Moreover, manipulation of Ipf1-mediated gene expression, such asof the insulin gene, may be useful for reshaping/repairing pancreatictissue both in vivo and in vitro. In one embodiment, the presentinvention makes use of the apparent involvement of the subjectIpf1protein in regulating the development of pancreatic tissueresponsible for formation of β-cells, e.g. induction of β-celldifferentiation from ductal tissue, as well as other tissue from thelungs and other organs which derive from the primitive gut. For example,therapeutic compositions for modulating the role of Ipf1 in tissuedifferentiation can be utilized to preserve any β-cells that have notbeen destroyed by diabetic or tumorogenic causes, as well as to induceregeneration of β-cells so as to increase the islet mass. In general,the subject method can be employed therapeutically to regulate thepancreas after physical, chemical or pathological insult.

[0079] In yet another embodiment, the subject method can be applied toto cell culture techniques, and in particular, may be employed toenhance the initial generation of prosthetic pancreatic tissue devices.Manipulation of Ipf1 function, for example, by altering the ability ofthe protein to transactivate Ipf1 responsive genes, can provide a meansfor more carefully controlling the characteristics of a cultured tissue.In an exemplary embodiment, the subject method can be used to augmentproduction of prosthetic devices which require 0-islet cells, such asmay be used in the encapsulation devices described in, for example, theAebischer et al. U.S. Pat. No. 4,892,538, the Aebischer et al. U.S. Pat.No. 5,106,627, the Lim U.S. Pat. No. 4,391,909, and the Sefton U.S. Pat.No. 4,353,888. Early progenitor cells to the pancreatic islets aremultipotential, and apparently coactive all the islet-specific genesfrom the time they first appear. As development proceeds, expression ofislet-specific hormones, such as insulin, becomes restricted to thepattern of expression characteristic of mature islet cells. Thephenotype of mature islet cells, however, is not stable in culture, asreappearence of embyonal traits in mature β-cells can be observed. Byutilizing agents which potentiate the action of Ipf1, such as Ipf1 geneexpression vectors. . .

[0080] Furthermore, manipulation of the differentiative state ofpancreatic tissue can be utilized in conjunction with transplantation ofartificial pancreas so as to promote implantation, vascularization, andin vivo differentiation and maintenance of the engrafted tissue. Forinstance, manipulation of Ipf1 function to affect tissue differentiationcan be utilized as a means of maintaining graft viability.

[0081] As set out above, the present method is also applicable to cellculture techniques. In one embodiment, manipulation of differentiativestates of renal or urogenital tissue can be performed in order toprovide cells lines, especially primary cell lines, which maintain aparticular phenotype, such as cell lines which are derived from utericbud cells. In another embodiment, the differentiation of gondal tissuein culture, such as Sertoli cells, can be controlled by manipulation ofthe subject Ipf1.

[0082] Conversely, control of one or more of the functions of Ipf1 canbe accomplished to inhibit differentiation along certain pathways,particularly where uncommitted pluripotent stem cells are beingcultured, so that cultures can be manipulated along alternatedevelopmental pathways. Accordingly, manipulation of Ipf1 function bythe present method to culture stem cells can be to inducedifferentiation of the uncommitted progenitor and thereby give rise to acommitted progenitor cell, or to cause further restriction of thedevelopmental fate of a committed progenitor cell towards becoming aterminally-differentiated neuronal cell. Such neuronal cultures can beused as convenient assay systems as well as sources of implantable cellsfor therapeutic treatments.

[0083] The manipulation of the biological function of the subject Ipf1can be carried out using solely such reagents as described herein, or incombination with treatment with neurotrophic factors which act to moreparticularly enhance a specific differentiation fate of the neuronalprogenitor cell. In the later instance, manipulation of Ipf1 involvementin cell regulation might be viewed as ensuring that the treated cell ispoised along a certain developmental pathway so as to be properlyinduced upon contact with a neurotrophic factor.

[0084] Another aspect of the present invention concerns recombinantforms of the subject Ipf1 polypeptides. The term “recombinant protein”refers to a protein of the present invention which is produced byrecombinant DNA techniques, wherein generally DNA encoding the subjectIpf1 protein is inserted into a suitable expression vector which is inturn used to transform a host cell to produce the heterologous protein.Moreover, the phrase “derived from”, with respect to a recombinant gene,is meant to include within the meaning of “recombinant protein” thoseproteins having an amino acid sequence of a native Ipf1, or an aminoacid sequence similar thereto which is generated by mutations includingsubstitutions and deletions (including truncation). Recombinant proteinspreferred by the present invention, in addition to native Ipf1, are atleast 60% homologous, more preferably 70% homologous and most preferably80% homologous with an amino acid sequence shown in one of SEQ ID No:2.Polypeptides having an activity of the subject Ipf1 polypeptides (i.e.either agonistic or antagonistic of the naturally-occurring protein) andhaving at least about 90%, more preferably at least about 95%, and mostpreferably at least about 98-99% homology with a sequence of either inSEQ ID No:2 are also within the scope of the invention.

[0085] The present invention further pertains to recombinant forms ofthe subject Ipf1 which are evolutionarily related to the Ipf1 proteinrepresented in SEQ ID No:2, that is, not identical, yet which arecapable of functioning as an agonist or an antagonist of at least onebiological activity of that protein. The term “evolutionarily relatedto”, with respect to amino acid sequences of recombinant Ipf1, refers toproteins which have amino acid sequences that have arisen naturally, aswell as to mutational variants which are derived, for example, byrecombinant mutagenesis. Such evolutionarily derived Ipf1 preferred bythe present invention are at least 60% homologous, more preferably 70%homologous and most preferably 80% homologous with the amino acidsequence shown in SEQ ID No:2. Polypeptides having at least about 90%,more preferably at least about 95%, and most preferably at least about98-99% homology with a sequence shown in SEQ ID No:2 are also within thescope of the invention.

[0086] The present invention further pertains to methods of producingthe subject Ipf1polypeptides. For example, a host cell transfected witha nucleic acid vector directing expression of Ipf1 can be cultured underappropriate conditions to allow expression of the polypeptide to occur.The polypeptide may be secreted, e.g. with the use of an exogenoussignal sequence, and isolated from a mixture of cells and mediumcontaining the recombinant protein. Alternatively, the peptide may beretained cytoplasmically, as the naturally occurring form of the proteinis believed to be, and the cells harvested, lysed and the proteinisolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The recombinant Ipf1 polypeptide can be isolated from cell culturemedium, host cells, or both using techniques known in the art forpurifying proteins including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, and immunoaffinitypurification with antibodies specific for such peptide. In a preferredembodiment, the recombinant Ipf1 is a fusion protein containing a domainwhich facilitates its purification, such as a glutathione-S-transferasedomain or a polyhistidine leader sequence in the form of a fusionprotein with the subject polypeptides.

[0087] This invention also pertains to a host cell transfected with anIpf1 gene in order to cause expression of a recombinant form of Ipf1.The host cell may be any prokaryotic or eukaryotic cell. Thus, anucleotide sequence derived from the cloning of the Ipf1 encoding all ora selected portion of the protein, can be used to produce a recombinantform of Ipf1 via microbial or eukaryotic cellular processes. Ligating apolynucleotide sequence into a gene construct, such as an expressionvector, and transforming or transfecting host cells with the vector arestandard procedures used in producing other well-known proteins, e.g.insulin, interferons, myc, p53, fos, jun, cyclins, Ikaros, and the like.Similar procedures, or modifications thereof, can be employed to preparerecombinant Ipf1, or portions thereof, by microbial means ortissue-culture technology in accord with the subject invention. Hostcells suitable for expression of recombinant Ipf1 polypeptides can beselected, for example, from amongst eukaryotic (yeast, avian, insect ormammalian) or prokaryotic (bacterial) cells.

[0088] The recombinant Ipf1 gene can be produced by ligating nucleicacid encoding a Ipf1, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells, or both.Expression vectors for production of recombinant forms of Ipf1includeplasmids and other vectors. For instance, suitable vectors for theexpression of Ipf1include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

[0089] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, andYRP17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused.

[0090] Preferred mammalian expression vectors contain prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription regulatory sequences that causeexpression bf a recombinant Ipf1 gene in eukaryotic cells. ThepcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG. pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found above inthe description of gene therapy delivery systems.

[0091] In some instances, it may be desirable to express a recombinantIpf1 by the use of a baculovirus expression system (see, for example,Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley &Sons: 1992). Examples of such baculovirus expression systems includepVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derivedvectors (such as pAcUW1), and pBlueBac-derived vectors (such as theβ-gal containing pBlueBac III).

[0092] The various methods employed in the preparation of the plasmidsand transformation of host organisms are well known in the art. Forother suitable expression systems for both prokaryotic and eukaryoticcells, as well as general recombinant procedures, see Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

[0093] When expression of a portion of one an Ipf1 protein is desired,i.e. a trunction mutant, it may be necessary to add a start codon (ATG)to the oligonucleotide fragment containing the desired sequence to beexpressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al. (1987) J Bacteriol. 169:751-XX57) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing Ipf1-derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al., supra).

[0094] Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene. This type of expression systemcan be useful under conditions where it is desirable to produce animmunogenic fragment of an Ipf1 protein. For example, the VP6 capsidprotein of rotavirus can be used as an immunologic carrier protein forportions of the Ipf1 polypeptide, either in the monomeric form or in theform of a viral particle. The nucleic acid sequences corresponding tothe portion of Ipf1 to which antibodies are to be raised can beincorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising a portionof the protein Ipf1 as part of the virion. It has been demonstrated withthe use of immunogenic fusion proteins utilizing the Hepatitis B surfaceantigen fusion proteins that recombinant Hepatitis B virions can beutilized in this role as well. Similarly, chimeric constructs coding forfusion proteins containing a portion of Ipf1and the poliovirus capsidprotein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see. for example, EP Publication No:0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

[0095] The Multiple Antigen Peptides System for peptide-basedimmunization can also be utilized to generate an immunogen, wherein adesired portion of Ipf1 is obtained directly from organo-chemicalsynthesis of the peptide onto an oligomeric branching lysine core (see,for example, Posnett et al. (1988) JBC 263:1719 and Nardelli et al.(1992) J. Immunol. 148:914). Antigenic determinants of the subjectIpf1-binding proteins can also be expressed and presented by bacterialcells.

[0096] In addition to utilizing fusion proteins to enhanceimmunogenicity, it is widely appreciated that fusion proteins can alsofacilitate the expression and purification of proteins, such the Ipf1polypeptides of the present invention. For example, Ipf1 can begenerated as a glutathione-S-transferase (GST) fusion protein. Such GSTfusion proteins can simplify purification of the recombinant protein, asfor example, by affinity pruification using glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausabel et al. (N.Y.: John Wiley & Sons, 1991)). In another embodiment,a fusion gene coding for a purification leader sequence, such as apeptide leader sequence comprising a poly-(His)/enterokinase cleavagesequence, can be added to the N-terminus of the desired portion of anIpf1 polypeptide in order to permit purification of the poly(His)-fusionprotein by affinity chromatography using a Ni²⁺ metal resin. Thepurification leader sequence can then be subsequently removed bytreatment with enterokinase (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

[0097] Techniques for making fusion genes are known to those skilled inthe art. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which are subsequently annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

[0098] The present invention also makes available isolated Ipf1polypeptides which are isolated from, or otherwise substantially free ofother cellular proteins, especially IEF1, IEF2, Isl-1 or othertranscriptional regulatory factors which might be associated with Ipf1or which bind nucleic acid containing Ipf1-responsive elements. The term“substantially free of other cellular or viral proteins” (also referredto herein as “contaminating proteins”) or “substantially pure orpurified preparations” are defined as encompassing preparations ofIpf1polypeptides having less than 20% (by. dry weight) contaminatingprotein, and preferably having less than 5% contaminating protein.Functional forms of the subject Ipf1polypeptides can be prepared, forthe first time, as purified preparations by providing recombinantproteins as described herein. By “purified”, it is meant, when referringto a polypeptide or DNA or RNA sequence, that the indicated molecule ispresent in the substantial absence of other biological macromolecules,such as other proteins (particularly transcriptional factors, as well asother contaminating proteins). The term “purified” as used hereinpreferably means at least 80% by dry weight, more preferably in therange of 95-99% by weight, and most preferably at least 99.8% by weight,of biological macromolecules of the same type present (but water,buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). The term “pure” asused herein preferably has the same numerical limits as “purified”immediately above. “Isolated” and “purified” do not encompass eithernatural materials in their native state or natural materials that havebeen separated into components (e.g., in an acrylamide gel) but notobtained either as pure (e.g. lacking contaminating proteins, orchromatography reagents such as denaturing agents and polymers, e.g.acrylamide or agarose) substances or solutions. Moreover, in theinstance of purified Ipf1, the protein preparation lacks anycontaminating nucleic acids, especially nucleic acid comprising a P1promoter sequence.

[0099] Furthermore, isolated peptidyl portions of full length forms ofIpf1 proteins can also be obtained by screening peptides recombinantlyproduced from the corresponding fragment of the nucleic acid encodingsuch peptides. In addition, fragments can be chemically synthesizedusing techniques known in the art such as conventional Merrifield solidphase f-Moc or t-Boc chemistry. Accordingly, DNA binding motifs (e.g.presumably inlcuding the homeodomain region) and activation domainswhich recruit other transcriptional factors (e.g. as may exist in theN-terminal fragment) can be refined to minimal sequences. For example,an Ipf1 protein may be arbitrarily divided into fragments of desiredlength with no overlap of the fragments, or preferably divided intooverlapping fragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as either agonists or antagonistsof wild-type Ipf1 activity. such as by microinjection assays or in vitroprotein or DNA binding assays. In an illustrative embodiment, peptidylportions of Ipf1, such as derived from the amino terminal half of theprotein or from the C-terminal portion, can tested for their ability toinhibit authentic Ipf1 activity by expression as thioredoxin fusionproteins, each of which contains a discrete fragment of the Ipf1 (see,for example, U.S. Pat. No. 5,270,181 and 5,292.646; and PCT publicationWO94/02502, as well the THIOFUSION kit of Invitrogen Inc, San Diego).Such fusion proteins can be utilized in the drug screening assaysdescribed below, and, if desired, peptidyl portions which areantagnositc can be synthesized as non-peptide analogs (e.g.,peptidomimetics).

[0100] It will also be possible to modify the structure of an Ipf1polypeptide for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified peptides, when designedto retain at least one activity of the naturally-occurring form of theprotein, are considered functional equivalents of the Ipf1 polypeptidesdescribed in more detail herein. Such modified peptide can be produced,for instance, by amino acid substitution, deletion, or addition.

[0101] For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. conservativemutations) will not have a major effect on the folding of the protein,and may or may not have much of an effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids are can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine.Phenylalanine, a tryptophan, and tyrosine are sometimes classifiedjointly as aromatic amino acids. In similar fashion, the amino acidrepertoire can be grouped as (1) acidic aspartate, glutamate; (2)basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine,valine, leucine, isoleucine, serine, threonine, with serine andthreonine optionally be grouped separately as aliphatic-hydroxyl; (4)aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine,glutamine; and (6) sulfur-containing=cysteine and methionine (see, forexample, Biochemistry, 2nd ed., Ed. by L. Stryer, WH Freeman and Co.:1981). Alternatively, amino acid replacement can be based on stericcriteria, e.g. isosteric replacements, without regard for polarity orcharge of amino acid sidechains. Whether a change in the amino acidsequence of a polypeptide results in a functional Ipf1 homolog (e.g.functional in the sense that it acts to mimic or antagonize thewild-type form) can be readily determined by assessing the ability ofthe variant peptide to produce a response in cells in a fashion similarto the wild-type Ipf1 protein or competitively inhibit such a response.Peptides in which more than one replacement has taken place can readilybe tested in the same manner.

[0102] This invention further contemplates a method of generating setsof combinatorial mutants of Ipf1, as well as truncation andfragmentation mutants, and is especially useful for identifyingpotential variant sequences (e.g. Ipf1 homologs) which are functional inIpf1-dependent transcriptional activation, but differ from a wild-typeform of the protein by, for instance, efficacy, potency and/orintracellular half-life. One purpose for screening such combinatoriallibraries is, for example, to isolate novel Ipf1 homologs which functionas either an agonist or an antagonist of the biological activities ofthe wild-type protein, or alternatively, possess novel activities alltogether. To illustrate, Ipf1 homologs can be engineered by the presentmethod to provide proteins which bind to Ipf1-responsive elements yetprevent complete assembly of Ipf1-dependent transcription regulatorycomplexes. Such proteins, when expressed from recombinant DNAconstructs, can be used in gene therapy protocols as Ipf1 antagonists.

[0103] Likewise, mutagenesis can give rise to Ipf1 homologs which haveintracellular half-lives dramatically different than the correspondingwild-type protein. For example, the altered protein can be renderedeither more stable or less stable to proteolytic degradation or othercellular process which result in destruction of, or otherwiseinactivation of, the naturally-occurring forms of Ipf1. Such Ipf1homologs, and the genes which encode them, can be utilized to alter theenvelope of expression for a particular recombinant Ipf1 by modulatingthe half-life of the recombinant protein. For instance, a shorthalf-life can give rise to more transient biological effects associatedwith a particular recombinant Ipf1 protein and, when part of aninducible expression system, can allow tighter control of recombinantprotein levels within a cell. As above, such proteins, and particularlytheir recombinant nucleic acid constructs, can be used in gene therapyprotocols.

[0104] In an illustrative embodiment of this method, the amino acidsequences for a population of Ipf1 homologs or other related proteinsare aligned, preferably to promote the highest homology possible. Such apopulation of variants can include, for example, Ipf1homologs from oneor more species (e.g. orthologs), or Ipf1 homologs from the same speciesbut which differ due to mutation, and other proteins related in some wayto Ipf1. Amino acids which appear at each position of the alignedsequences are selected to create a degenerate set of combinatorialsequences. There are many ways by which the library of potential Ipf1homologs can be generated from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then be ligatedinto an appropriate gene for expression. The purpose of a degenerate setof genes is to provide, in one mixture, all of the sequences encodingthe desired set of potential Ipf1 sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierpp. 273-289: Itakura et al. (1984) Annu. Rev. Biocheem. 53.323; Itakuraet al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.11:477. Such techniques have been employed in the directed evolution ofother proteins (see, for example, Scott et al. (1990) Science249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al.(1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

[0105] Alternatively, other forms of mutagenesis can be utilized togenerate a combinatorial library. For example, Ipf1 homologs (bothagonist and antagonist forms) can be generated and isolated from alibrary by screening using, for example, alanine scanning mutagenesisand the like (Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al.(1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene137:109-118; Grodberg et al. (1993) Eur. J Biochem. 218:597-601;Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892; Lowman et al.(1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science244:1081-1085), by linker scanning mutagenesis (Gustin et al. (1993)Virology 193:653-660; Brown et al. (1992) Mol. Cell Biol. 12:2644-2652;McKnight et al. (1982) Science 232:316); by saturation mutagenesis(Meyers et al. (1986) Science 232:613); by PCR mutagenesis (Leung et al.(1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis (Milleret al. (1992) A Short Course in Bacterial Genetics, CSHL Press, ColdSpring Harbor, N.Y.; and Greener et al. (1994) Strategies in Mol Biol7:32-34).

[0106] A wide range of techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations, aswell as for screening cDNA libraries for gene products having a certainproperty. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of Ipf1. The most widely used techniques for screening largegene libraries typically comprises cloning the gene library intoreplicable expression vectors, transforming appropriate cells with theresulting library of vectors, and expressing the combinatorial genesunder conditions in which detection of a desired activity facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected. The illustrative assays described below are amenable tohigh through-put analysis as necessary to screen large numbers ofdegenerate Ipf1 sequences created by combinatorial mutagenesistechniques.

[0107] In one screening assay, the candidate gene products are expressedin cells which are co-transfected within Ipf1-dependent reporterconstruct, such as the Ins-CAT vector described in Walker et al. (1983,Nature 306:557-561). Ipf1 homologs from the library which can mimic thefunction of Ipf1 (e.g. or Ipf1 agonists) will be detected by theirability to activate expression of the reporter gene. In preferredembodiments, detection and isolation of genes encoding Ipf1 agonistsutilize a reporter construct which permits isolation of cells expressingthese genes by providing a selectable marker such as drug resistance orluminescence. For example, a reporter construct can be provided whichplaces the neo gene (provides resistance to G418 antibiotics) under thecontrol of an Ipf1-responsive element, such as multiple P1 promotersequences. Agonistic forms of Ipf1 will therefor confer resistance toG418, and permit isolation of Ipf1 clones from the library based on thatselection criteria. Alternatively, the drug resistance gene can bereplaced with a luminescence marker such as luciferase, such thatIpf1-induced expression is detected by luminescence of the cell.Accordingly, Ipf1 clones which activate expression of the luminescencemarker can be isolated from the library by, for example, sorting thetransfected cells with a fluorescence-activated cell sorter (FACS).

[0108] In similar fashion, antagonistic mutants of Ipf1 can be detectedand isolated from the library based on their ability inhibit Ipf1activation of a reporter gene. Co-transfection of cells with theconstructs of the Ipf1 library, wild-type Ipf1, and a reporter genepermit this inhibitory activity to be observed. For example, theluciferase reporter described above, when transfected in a cellexpressing wild-type Ipf1 and an Ipf1 mutant from the library, will beactivated in cells wherein the Ipf1 mutant is an agonist, ordysfunctional (e.g. mis-folded), but repressed whenever an Ipf1 mutantantagonizes the function of the wild-type Ipf1 protein. For instance,Ipf1 homologs can be isolated from the library which the retain theability to bind an Ipf1-responsive element, but which are defective forrecruiting other transcriptional complexes to the promoter site, oralternatively, which retain the ability to bind other proteins involvedin Ipf1 complexes but which are defective in binding to anIpf1-responsive element. The reporter construct may also be generatedwith a marker gene whose expression is toxic or cytostatic to the hostcell such that expression of an Ipf1 antagonist is detected by itsability to rescue the cell through inhibition of the reporter geneexpression.

[0109] The invention also provides for reduction of the Ipf1 protein togenerate mimetics, e.g. peptide or non-peptide agents, which are able todisrupt binding of Ipf1 to promoter sequences or to other regulatoryproteins. Thus, such mutagenic techniques as described above are alsouseful to map the determinants of Ipf1 which participate inprotein-protein interactions involved in, for example, formingtranscriptional complexes. To illustrate, the critical residues of aIpf1 which are involved in molecular recognition of Ipf1 can bedetermined and used to generate Ipf1-derived peptidomimetics thatcompetitively inhibit binding of Ipf1 to other regulatory proteins or toIpf1-responsive elements. By employing, for example, scanningmutagenesis to map the amino acid residues of Ipf1 apparently involvedin complex formation, peptidomimetic compounds can be generated whichmimic those residues, and which, by inhibiting binding of the Ipf1 toother regulatory proteins, can interfere with the function of Ipf1 intranscriptional regulation of one or more genes. For instance,non-hydrolyzable peptide analogs of such residues can be generated usingretro-inverse peptides (e.g. see U.S. Pat. Nos. 5,116,947 and 5,218,089;and Pallai et al. (1983) Int J Pept Protein Res 21:84-92) benzodiazepine(e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands. 1988), azepine(e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substitutedgama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295;and Ewenson et al. in Peptides: Structure and Function (Proceedings ofthe 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill.,1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), andβ-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71).

[0110] Another aspect of the invention pertains to an antibodyspecifically reactive with an Ipf1 protein. For example, by usingimmunogens derived from Ipf7, anti-protein/anti-peptide antisera ormonoclonal antibodies can be made by standard protocols (See, forexample, Antibodies: A Laboratory Manual ed. by Harlow and Lane (ColdSpring Harbor Pros: 1988)). A mammal, such as a mouse, a hamster orrabbit can be immunized with an immunogenic form of the peptide (e.g., afull length Ipf1 or an antigenic fragment which is capable of elicitingan antibody response). Techniques for conferring immunogenicity on aprotein or peptide include conjugation to carriers or other techniqueswell known in the art. An immunogenic portion of Ipf1 can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies. In a preferred embodiment,the subject antibodies are immunospecific for antigenic determinants ofan Ipf1 protein of the present invention, e.g. antigenic determinants ofthe protein represented by SEQ ID No:2 or a closely related human ornon-human mammalian homolog thereof. For instance, a favoredanti-Ipf1antibody of the present invention does not substantially crossreact (i.e. react specifically) with a protein which is less than 90percent homologous to SEQ ID No:2; though antibodies which do notsubstantially cross react with a protein which is less than 95 percenthomologous with SEQ ID No:2, or even less than 98-99 percent homologouswith SEQ ID No:2, are specifically contemplated. By “not substantiallycross react”, it is meant that the antibody has a binding affinity for anon-homologous protein (e.g. other insulin promoter-binding proteinssuch as IEF2, as well as other homeobox proteins which do not bind theinsulin promoter) which is at least one order of magnitude, morepreferably at least two orders of magnitude and even more preferably atleast 3 orders of magnitude less than the binding affinity for a proteinrepresented by SEQ ID No:2.

[0111] Following immunization, anti-Ipf1 antisera can be obtained and.if desired, polyclonal anti-Ipf1 antibodies isolated from the serum. Toproduce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a Ipf1 of thepresent invention and monoclonal antibodies isolated from a culturecomprising such hybridoma cells.

[0112] The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with an Ipf1 protein.Antibodies can be fragmented using conventional techniques, includingrecombinant engineering, and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments. The antibody of the present invention is furtherintended to include bispecific and chimeric molecules having ananti-Ipf1 portion.

[0113] Both monoclonal and polyclonal antibodies (Ab) directed againstan Ipf1 protein can be used to block the action of that protein andallow the study of the role of Ipf1 in transcriptional regulationgenerally, or in the etiology of β-cell development or islet celltransformation, e.g. by microinjection of anti-Ipf1 into cells.

[0114] Antibodies which specifically bind Ipf1 epitopes can also be usedin immunohistochemical staining of tissue samples in order to evaluatethe abundance and pattern of expression of Ipf1. Anti-Ipf1 antibodiescan be used diagnostically in immuno-precipitation and immuno-blottingto detect and evaluate Ipf1 levels in tissue or bodily fluid as part ofa clinical testing procedure. For instance, such measurements can beuseful in predictive valuations of the onset or progression of, forexample, diabetes or other β-cell abnormalities Likewise, the ability tomonitor Ipf1 levels in the cells of an individual can permitdetermination of the efficacy of a given treatment regimen for anindividual afflicted with such a disorder. The level of Ipf1 can bemeasured in cells found in bodily fluid or can be measured in tissue,such as pancreatic biopsies. Diagnostic assays using anti-Ipf1antibodiescan include, for example, immunoassays designed to aid in earlydetection of β-cell necrosis (e.g. IDPM), or in the diagnosis of aneoplastic or hyperplastic disorder, and may aid in detecting thepresence by detecting cells in which a lesion of the Ipf1 gene hasoccurred or in which the protein is misexpressed or found in abnormalprotein complexes, or found in abnormally high levels in serum or plasmaindicating cytolysis of β-cells.

[0115] Another application of the subject antibodies is in theimmunological screening of cDNA libraries constructed in expressionvectors such as λgt11, λct18-23, λZAP, and λORF8. Messenger libraries ofthis type, having coding sequences inserted in the correct reading frameand orientation, can produce fusion proteins. For instance, λgt11 willproduce fusion proteins whose amino termini consist of β-galactosidaseamino acid sequences and whose carboxy termini consist of a foreignpolypeptide. Antigenic epitopes of an Ipf1protein can then be detectedwith antibodies, as, for example, reacting nitrocellulose filters liftedfrom infected plates with anti-Ipf1 antibodies. Phage, scored by thisassay, can then be isolated from the infected plate. Thus, the presenceof Ipf1 homologs can be detected and cloned from other animals,including humans, and alternate isoforms (including splicing variants)can also be detected and cloned from the same species.

[0116] Moreover, the nucleotide sequence determined from the cloning ofthe subject Ipf1will further allow for the generation of probes designedfor use in identifying homologs in other cell types, as well as Ipf1homologs (e.g. orthologs) from other animals. For instance, the presentinvention also provides a probe/primer comprising a substantiallypurified oligonucleotide, which oligonucleotide comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast 10 consecutive nucleotides of sense or anti-sense sequence of SEQID No:1, or naturally occurring mutants thereof. In preferredembodiments, the probe/primer further comprises a label group attachedthereto and able to be detected, e.g. the label group is selected fromthe group consisting of radioisotopes, fluorescent compounds, enzymes,and enzyme co-factors. Such probes can also be used as a part of adiagnostic test kit for identifying transformed cells, such as formeasuring a level of an Ipf1 nucleic acid in a sample of cells from apatient; e.g. detecting mRNA encoding Ipf1mRNA level or determiningwhether a genomic Ipf1 gene has been mutated or deleted.

[0117] In addition, nucleotide probes can be generated which allow forhistological screening of intact tissue and tissue samples for thepresence of an Ipf1 mRNA. Similar to the diagnostic uses of anti-Ipf7antibodies, the use of probes directed to Ipf1 mRNAs, or to genomic Ipf1sequences, can be used for both predictive and therapeutic evaluation ofallelic mutations which might be manifest in, for example, diabeticdisorders as well as neoplastic or hyperplastic disorders (e.g. unwantedcell growth) or abnormal differentiation of tissue. Used in conjunctionwith an antibody immunoassays, the nucleotide probes can help facilitatethe determination of the molecular basis for a developmental disorderwhich may involve some abnormality associated with expression (or lackthereof) of Ipf1. For instance, variation in synthesis of Ipf1 can bedistinguished from a mutation in the genes coding sequence.

[0118] Accordingly, the present method provides a method for determiningif a subject is at risk for a disorder characterized by unwanted cellproliferation or abherent control of differentiation, particularly ofpancreatic tissue. In preferred embodiments, the subject method can begenerally characterized as comprising detecting, in a tissue sample ofthe subject (e.g. a human patient), the presence or absence of a geneticlesion characterized by at least one of (i) a mutation of a geneencoding Ipf1 or (ii) the mis-expression of an Ipf1 gene. To illustrate,such genetic lesions can be detected by ascertaining the existence of atleast one of (i) a deletion of one or more nucleotides from an Ipf1gene, (ii) an addition of one or more nucleotides to such an Ipf1 gene,(iii) a substitution of one or more nucleotides of an Ipf1 gene, (iv) agross chromosomal rearrangement of an Ipf1 gene, (v) a gross alterationin the level of a messenger RNA transcript of an Ipf1 gene, (vi) thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of an Ipf1 gene, and (vii) a non-wild type level of an Ipf1protein. In one aspect of the invention there is provided a probe/primercomprising an oligonucleotide containing a region of nucleotide sequencewhich is capable of hybridizing to a sense or antisense sequence of SEQID No:1, or naturally occurring mutants thereof, or 5′ or 3′ flankingsequences or intronic sequences naturally associated with the subjectIpf1 gene. The probe is exposed to nucleic acid of a tissue sample; andthe hybridization of the probe to the sample nucleic acid is detected.In certain embodiments, detection of the lesion comprises utilizing theprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202) or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al. (1988) Science,241:1077-1080; and NaKazawa et al. (1944) PNAS 91:360-364) the later ofwhich can be particularly useful for detecting point mutations in anIpf1 gene. Alternatively, immunoassays can be employed to determine thelevel of Ipf1 protein and/or its participation in protein complexes,particularly transcriptional regulatory complexes such as those whichactivate insulin expression.

[0119] Also, by inhibiting endogenous production of Ipf1, anti-sensetechniques (e.g. microinjection of antisense molecules, or transfectionwith plasmids whose transcripts are anti-sense with regard to an Ipf1mRNA or gene sequence) can be used to investigate the role of Ipf1 ingrowth and differentiative events, such as those giving rise topancreatic development, as well as abnormal cellular functions in whichIpf1 may participate, e.g. in mis-regulation of insulin expression. Suchtechniques can be utilized in cell culture, but can also be used in thecreation of transgenic animals.

[0120] Furthermore, by making available purified and recombinant Ipf1,the present invention facilitates the development of assays which can beused to screen for drugs which are either agonists or antagonists of thecellular function Ipf1, such as its role in the pathogenesis ofproliferative and differentiative disorders, as well as in insulinregulation. For instance, an assay can be generated according to thepresent invention which evaluates the ability of a compound to modulatebinding between Ipf1 and other transcriptional regulatory proteins orIpf1-responsive elements. A variety of assay formats will suffice and,in light of the present inventions, will be comprehended by skilledartisan.

[0121] In many drug screening programs which test libraries of compoundsand natural extracts. high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target when contacted with a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with other proteinswith a nucleic acid. Accordingly, in an exemplary screening assay of thepresent invention, the compound of interest is contacted with a mixturegenerated from an isolated and purified Ipf1 polypeptide and a nucleicacid which specifically binds Ipf1 (e.g. Ipf1-responsive element) suchas the P1 insulin promoter. Detection and quantification ofIpf1/promoter complexes provides a means for determining the compound'sefficacy at inhibiting (or potentiating) DNA binding by Ipf1. Similarly,other regulatory proteins which are identified as binding Ipf1 can beused in place of the nucleic acid. The efficacy of the compound can beassessed by generating dose response curves from data obtained usingvarious concentrations of the test compound. Moreover, a control assaycan also be performed to provide a baseline for comparison. In thecontrol assay, isolated and purified Ipf1 is added to a compositioncontaining the nucleic acid (or the other regulatory proteins), and theformation of Ipf1-containing complexes is quantitated in the absence ofthe test compound.

[0122] The formation of complexes including Ipf1 may be detected by avariety of techniques. For instance, modulation in the formation ofcomplexes can be quantitated using for example, detectably labelledproteins (e.g. radiolabelled, fluorescently labelled, or enzymaticallylabelled), by immunoassay, or by chromatographic detection.

[0123] Typically, it will be desirable to immobilize either the Ipf1protein or the DNA or other regulatory protein (hereinafter “targetmolecule”) to facilitate separation of target/Ipf1complexes fromuncomplexed forms, as well as to accomadate automation of the assay. Inan illustrative embodiment, a fusion protein can be provided which addsa domain that permits Ipf1 to be bound to an insoluble matrix. Forexample, glutathione-S-transferase/Ipf1 (GST/Ipf1) fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the target molecule, e.g. an ³⁵S-labeled protein or DNAfragment, and the test compound, and mixture incubated under conditionsconducive to complex formation. Following incubation, the beads arewashed to remove any unbound target molecule and the matrix bead-boundradiolabel determined directly (e.g. beads placed in scintilant), or inthe superntantant after the complexes are dissociated, e.g. whenmicrotitre plates are used. Alternatively, after washing away unboundprotein, the complexes can be dissociated from the matrix, separated bySDS-PAGE gel, and the amount of target molecules found in thematrix-bound fraction quantitated from the gel using standardelectrophoretic techniques.

[0124] Other techniques for immobilizing proteins or DNA on matrices arealso available for use in the subject assay. For instance, the DNAtarget protein can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated DNA can be prepared using techniques wellknown in the art and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical) and Ipf1 binding to the immobilizednucleic acid detected. Exemplary methods for detecting such complexes,in addition to those described above for the GST-immobilized Ipf1complexes, include immunodetection of complexes using antibodiesreactive with Ipf1 as well as enzyme-linked assays which rely ondetecting an enzymatic activity associated with Ipf1. In the instance ofthe latter, the enzyme can be chemicaly conjugated or provided as afusion protein with the Ipf1 polypeptide. To illustrate, Ipf1 can bechemically cross-linked with alkaline phosphatase, and the amount ofIpf1 trapped in the complex can be assessed with a chromogenic substrateof the enzyme, e.g. paranitrophenyl phosphate. Likewise, a fusionprotein comprising the Ipf1 and glutathione-S-transferase can beprovided, and complex formation quantitated by detecting the GSTactivity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J. BiolChem 249:7130).

[0125] For processes which rely on immunodetection for quatitating Ipf1trapped in the complex, antibodies against the protein, such as theanti-Ipf1 antibodies described herein, can be used. Alternatively, theprotein to be detected in the complex can be “epitope tagged” in theform of a fusion protein which includes, in addition to Ipf1 sequences,a second polypeptide for which antibodies are readily available (e.g.from commercial sources). For instance, the GST fusion proteinsdescribed above can also be used for quantification of binding usingantibodies against the GST moiety. Other useful epitope tags includemyc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem266:21150-21157) which includes a 10-residue sequence from c-myc, aswell as the pFLAG system (International Biotechnologies, Inc.) or thepEZZ-protein A system (Pharamacia, NJ).

[0126] In another embodiment, the assay format is derived in a similarmanner to the use of Ipf1-sensitve reporter constructs described above.For example, co-transfection of an Ipf1-deficient cell (e.g. a COS orCHO cell) with an Ipf1 expression vector and an Ipf1-dependent reporterconstruct provides a convenient system for identifying compounds basedon their ability to affect Ipf1-dependent transcription.

[0127] Additionally, Ipf1 can be used to generate an interaction trapassay (see, U.S. Pat. No. 5,283,317: PCT publication WO94/10300; Zervoset al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696), for detecting agents whicheither potentiate or attenuate complex formation between a Ipf1 andother transcriptional regulatory proteins. Indeed, an interaction trapassay generated with Ipf1 as a bait protein can be used to identifyother cellular proteins which bind Ipf1, and which would therefore beimplicated in Ipf1 transcriptional regulation, such as by participatingin regulatory complexes, or by causing post-translational modification(e.g. phosphenylation or ubiquitination) of Ipf1.

[0128] The interaction trap assay relies on reconstituting in vivo afunctional transcriptional activator protein from two separate fusionproteins, one of which comprises the DNA-binding domain of atranscriptional activator fused to an Ipf1 polypeptide (which preferablylacks its own DNA binding ability). The second fusion protein comprisesa transcriptional activation domain (e.g. able to initiate RNApolymerase transcription) fused to a protein which binds Ipf1. When thetwo fusion proteins interact, the two domains of the transcriptionalactivator protein are brought into sufficient proximity as to causetranscription of a reporter gene. In an illustrative embodiment,Saccharomyces cerevisiae YPB2 cells are transformed simultaneously witha plasmid encoding a GAL4db-Ipf1 (Δ homeodomain) fusion (db: DNA bindingdomain) and with a plasmid encoding the GAL4 activation domain (GAL4ad)fused to an Ipf1-binding protein, wherein Ipf1 (Δ homeodomain)designates an Ipf1 mutant lacking a homeodomain able to anIpf1-responsive ement, such as an Ipf1 in which His-190 is deleted, orwherein the protein is truncated (e.g. comprises residues 1-145).Moreover, the strain is transformed such that the GAL4-responsivepromoter drives ILLS expression of a phenotypic marker. For example, theability to grow in the absence of histidine can depends on theexpression of the HIS3 gene. When the HIS3 gene is placed under thecontrol of a GAL4-responsive promoter, relief of this auxotrophicphenotype indicates that a functional GAL4 activator has beenreconstituted through the interaction of the target protein and Ipf1.Thus, agents able to inhibit Ipf1 interaction with target protein 20will result in yeast cells unable to growth in the absence of histidine.Alternatively, the phenotypic marker (e.g. instead of the HIS3 gene) canbe one which provides a negative selection when expressed such thatagents which disrupt this Ipf1-dependent interaction confer positivegrowth selection to the cells. Comercial kits which can be modified todevelop two-hybrid assays with the subject Ipf1 are presently available(e.g., MATCHMAKER kit, ClonTech catalog number K1605-1, Palo Alto,Calif.). This assay can also be used to screen cDNA libraries for Ipf1interactors, by generating a library of cDNA:Ad constructs.

[0129] Another aspect of the present invention concerns transgenicanimals which are comprised of cells (of that animal) which contain atransgene of the present invention and which preferably (thoughoptionally) express an exogenous Ipf1 in one or more cells in theanimal. The Ipf1 transgene can encode the wild-type form of the protein,or can encode homologs thereof. including both agonists and antagonists,as well as antisense constructs designed to inhibit expression of theendogenous gene. In preferred embodiments, the expression of thetransgene is restricted to specific subsets of cells, tissues ordevelopmental stages utilizing, for example, cis-acting sequences thatcontrol expression in the desired pattern. In the present invention,such mosiac expression of the subject Ipf1 can be essential for manyforms of lineage analysis and can additionally provide a means to assessthe effects of, for example, antagonism of Ipf1 action, which deficiencymight grossly alter development in small patches of tissue within anotherwise normal embryo. Toward this and, tissue-specific regulatorysequences and conditional regulatory sequences can be used to controlexpression of the transgene in certain spatial patterns. Moreover,temporal patterns of expression can be provided by, for example,conditional recombination systems or prokaryotic transcriptionalregulatory sequences.

[0130] Genetic techniques which allow for the expression of transgenescan be regulated via site-specific genetic manipulation in vivo areknown to those skilled in the art. For instance, genetic systems areavailable which allow for the regulated expression of a recombinase thatcatalyzes the genetic recombination a target sequence. As used herein,the phrase “target sequence” refers to a nucleotide sequence that isgenetically recombined by a recombinase. The target sequence is flankedby recombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of Ipf1 or in disruption of the coding Jib sequence. Forexample, excision of a target sequence which interferes with theexpression of a recombinent Ipf1 gene can be designed to activateexpression of that gene. This interference with expression of theprotein can result from a variety of mechanisms, such as spatialseparation of the gene from a promoter element or an internal stopcodon. Moreover, the transgene can be made wherein the coding sequenceof the gene is flanked by recombinase recognition sequences and isinitially transfected into cells in a 3′ to 5′ orientation with respectto the promoter element. In such an instance, inversion of the targetsequence will reorient the subject gene by placing the 5′ end of thecoding sequence in an orientation with respect to the promoter elementwhich allow for promoter driven transcriptional activation.Alternatively, recombinase sites can be placed in intronic sequence and,by homologous recombination inserted into the genomic Ipf1 gene suchthat inversion of excisim of the target sequence inactivates the Ipf1allele.

[0131] In an illustrative embodiment, either the cre/loxP recombinasesystem of bacteriophage Pi (Lakso et al. (1992) PNAS 89:6232-6236; Orbanet al. (1992) PNAS 89:6861-6865) or the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355;PCT publication WO 92/15694) can be used to generate in vivosite-specific genetic recombination systems. Cre recombinase catalyzesthe site-specific recombination of an intervening target sequencelocated between loxP sequences. loxP sequences are 34 base pairnucleotide repeat sequences to which the Cre recombinase binds and arerequired for Cre recombinase mediated genetic recombination. Theorientation of loxP sequences determines whether the intervening targetsequence is excised or inverted when Cre recombinase is present(Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing theexcision of the target sequence when the loxP sequences are oriented asdirect repeats and catalyzes inversion of the target sequence when loxPsequences are oriented as inverted repeats.

[0132] Accordingly, genetic recombination of the target sequence isdependent on expression of the Cre recombinase. Expression of therecombinase can be regulated by promoter elements which are subject toregulatory control, e.g., tissue-specific, developmental stage-specific,inducible or repressible by externally added agents. This regulatedcontrol will result in genetic recombination of the target sequence onlyin cells where recombinase expression is mediated by the promoterelement. Thus, the activation or exogenous expression of a Ipf1, oralternatively, disruption of the endogenous Ipf1 gene, can be regulatedvia regulation of recombinase expression.

[0133] Use of the cre/loxP recombinase system to regulate expression ofa recombinant Ipf1gene, requires the construction of a transgenic animalcontaining transgenes encoding both the Cre recombinase and the subjectprotein. Animals containing both the Cre recombinase and the recombinantIpf1 gene can be provided through the construction of “double”transgenic animals. A convenient method for providing such animals is tomate two transgenic animals each containing a transgene, e.g., the Ipf1gene in one animal and recombinase gene in the other. Similar transgenemanipulation can be used to generate animals dependent on recombinaseexpression for disruption of the Ipf1 gene.

[0134] One advantage derived from initially constructing transgenicanimals containing a transgene in a recombinase-mediated expressibleformat derives from the likelihood that the subject protein will bedeleterious upon expression in the transgenic animal such as thepancreas deficient mice described below. In such an instance, a founderpopulation, in which the subject transgene is silent in all tissues, canbe propagated and maintained. Individuals of this founder population canbe crossed with animals expressing the recombinase in, for example, oneor more tissues. Thus, the creation of a founder population in which,for example, an antagonistic Ipf1 transgene is silent will allow thestudy of progeney from that founder in which disruption of Ipf1transcriptional regulatory complexes in a particular tissue or atcertain developmental stages would result in, for example, a lethalphenotype.

[0135] Similar conditional transgenes can be provided using eitherprokaryotic or viral promoter sequences which require prokaryotic orviral proteins to be simultaneous expressed in the cell in order tofacilitate expression of the transgene. Exemplary promoters and thecorresponding trans-activating prokaryotic proteins are given in U.S.Pat. No. 4,833,080, and conditional viral expression systems areprovided in U.S. Pat. No. 5,221,778. Moreover, expression of theconditional transgenes can be induced by gene therapy-like methodswherein a gene encoding the trans-activating protein, e.g. a recombinaseor a prokaryotic protein, is delivered to the tissue and caused to beexpressed using, for example, one of the gene therapy constructsdescribed above. By this method, the Ipf1 transgene could remain silentinto adulthood and its expression “turned on” by the introduction of thetrans-activator.

[0136] Methods of making transgenic animals are well known in the art.For example, see Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986), and U.S. Pat. Nos.5,347,075; 5,322,775; 5,221,778; 5,175,385; 5,175,384; 5,175,383;5,087,571; and 4,736,866.

[0137] In an exemplary embodiment, the “transgenic non-human animals” ofthe invention are produced by introducing transgenes into the germlineof the non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2 pl of DNA solution. The useof zygotes as a target for gene transfer has a major advantage in thatin most cases the injected DNA will be incorporated into the host 15ggene before the first cleavage (Brinster et al. (1985) PNAS82:4438-4442). As a consequence, all cells of the transgenic non-humananimal will carry the incorporated transgene. This will in general alsobe reflected in the efficient transmission of the transgene to offspringof the founder since 50% of the germ cells will harbor the transgene.Microinjection of zygotes is the preferred method for incorporatingtransgenes in practicing the invention.

[0138] For construction of transgenic mice, procedures for embryomanipulation and microinjection are described in Hogan et al.Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. In an exemplary embodiment, mouse zygotesare collected from six week old females that have been superovulatedwith pregnant mares serum (PMS) followed 48 hours later with humanchorionic gonadotropin. Primed females are placed with males and checkedfor vaginal plugs on the following morning. Pseudopregnant females areselected for estrus, placed with proven sterile vasectomized males andused as recipients. Zygotes are collected and cumulus cells removed bytreatment with hyaluronidase (1 mg/ml). Pronuclear embryos are recoveredfrom female mice mated to males. Females are treated with pregnant mareserum, PMS (5 IU) to induce follicular growth and human chorionicgonadotropin, hCG (51 U) to induce ovulation. Embryos are recovered in aDulbecco's modified phosphate buffered saline (DPBS) and maintained inDulbecco's modified essential medium (DMEM) supplemented with 10% fetalbovine serum.

[0139] Microinjections can be performed, for example, using Narishigemicromanipulators attached to a Nikon diaphot microscope. Embryos areheld in 100 microliter drops of DPBS under oil while beingmicroinjected. DNA solution is microinjected into the largest visiblemale pronucleus. Successful injection is monitored by swelling of thepronucleus. Immediately after injection embryos are transferred torecipient females, mature mice mated to vasectomized male mice.Recipient females are anesthetized using 2,2,2-tribromoethanol.Paralumbar incisions are made to expose the oviducts and the embryos aretransformed into the ampullary region of the oviducts. The body wall issutured and the skin closed with wound clips. Recipients areappropriately ear notched for identification and maintained untilparturition.

[0140] To identify transgenic offspring, particularly where conditionaltransgenic systems have been employed such that no phenotypic trait isapparent absent induction, standard tail samples can be used to assessincorporation of the transgene. For example, at three weeks of age,about 2-3 cm long tail samples are excised for DNA analysis. The tailsamples are digested by incubating overnight at 55° C. in the presenceof 0.7 ml 50 mM Tris, pH 8.0, 100 mM EDTA, 0.5% SDS and 350 μg ofproteinase K. The digested material is extracted once with equal volumeof phenol and once with equal volume of phenol:chloroform (1:1 mixture).The supernatants are mixed with 70 μl 3 M sodium acetate (pH 6.0) andthe DNAs are precipitated by adding equal volume of 100% ethanol. TheDNAs are spun down in a microfuge, washed once with 70% ethanol, driedand dissolved in 100 μl TE buffer (10 mM Tris, pH 8.0 and 1 mM EDTA). 10to 20 μl of DNAs were cut with restrictions based on the transgene map,electrophoresed on agarose gels, blotted onto nitrocellulose paper andhybridized with ¹³P-labeled probes described herein.

[0141] Retroviral infection can also be used to introduce a Ipf1transgene into a non-human animal. The developing non-human embryo canbe cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retroviral infection (Jaenich, R. (1976)PNAS 73 :1260-1264). Efficient infection of the blastomeres is obtainedby enzymatic treatment to remove the zona pellucida (Manipulating theMouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1986). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al.(1985) PNAS 82:6148-6152). Transfection is easily and efficientlyobtained by culturing the blastomeres on a monolayer of virus-producingcells (Van der Putten, supra; Stewart et al. (1987) EMBO J 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

[0142] A third type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells are obtained from pre-implantationembryos cultured in vitro and fused with embryos (Evans et al. (1981)Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler etal. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

[0143] Methods of making knock-out or disruption transgenic animals arealso generally known. See, for example, Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).An exemplary knock-out mouse is described in the examples below. As setout above, recombinase-dependent knockouts can also be generated, e.g.by homologous recombination to insert recombinase target sequences, suchthat tissue specific and/or temporal control of inactivation of theendogenous Ipf1 gene can be controlled as above.

Exemplification

[0144] The invention now being generally described, it will be morereadily understood by reference to the following examples which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention, and are not intended to limit theinvention.

EXAMPLE 1 Cloning and Expression of Ipf1

[0145] As described below, a cDNA encoding Ipf1, a novel mammalianhomeodomain-containing protein, has been isolated. Ipf1 is apparentlyexpressed predominantly in the β-cells of normal adulst mouse pancreas,and it binds to and transactivates the insulin promoter, providingevidence that Ipf1 is directly involved in the selective β-cellexpression of the insulin gene. In mouse embryos, Ipf1 expression isinitiated prior to hormone gene expression and restricted to the ventraland dorsal walls of primative foregut at positions where pancreas willlater form. The pattern of Ipf1 expression and its ability to stimulateinsulin gene transcription suggests that Ipf1 functions both in theearly specification of the primative gut to a pancreatic fate and in thematuration of the pancreatic β-cell.

[0146] The transcriptional activity of the rat insulin I 5′ flanking DNAis to a large extent mediated by the enhancer element which containsbinding sites for a number of trans-acting nuclear proteins that eachcontribute to the overall activity of the enhancer (German et al.,(1992) Genes Dev 6:2165-2176). Although the enhancer element isdominant, it bas previously been shown that the proximal ‘promoter’sequences have a low intrinsic cell specific activity (Edlund et al.,(1985) Science 231:912-916). As described herein, mutation of the P1promoter site results in a 2.5-fold decrease in transcriptional activityof the whole insulin 5′ flank. It is also demonstrated that recombinantIpf1 binds to the P1 site and is capable of increasing the activity ofthe complete insulin 5′ flanking DNA in transiently transfectedinsulin-producing PTC1 cells and that this transactivation is dependenton the P1 promoter site. Ipf1 can also transactivate multimers of the P1site linked to a heterologous TATA box in non-βcells. The relatively lowdegree of transactivation by Ipf1 of the isolated P1 site probablyreflects the need for multiple transacting factors in thetranscriptional regulation of the insulin gene. Since Ipf1 is restrictedto the β-cells of adult pancreas and binds to and transactivates theinsulin promoter, it is very likely that Ipf1 in vivo contributes to theα-cell specific activity of the insulin promoter. Ipf1 may alsocontribute to other aspects of the β-cell phenotype.

[0147] In the mouse, morphogenesis of the pancreas begins by evaginationof the duodenum at the 26 somite stage (e9.5) (Gittes (1992) PNAS89:1128-1132, but the midgut and adjacent tissue of mouse embryosacquire the ability to form exocrine pancreas tissue in vitro at aboutthe 8 somite stage. By the 10 somite stage, a region of the gut itselfcan be identified as the precursor of exocrine pancreas (Wessells et al.(1967) Dev. Biol. 15:237-270). The onset of Ipf1 protein expression ataround the 13 somite stage supports a role for Ipf1 in the commitment ofthe primitive foregut endoderm to a pancreatic fate. At this early stageof development, no “pancreatic” mesoderm or even loose mesoderm isassociated with the dorsal gut endoderm which instead is in closeproximity to the notochord (Wessells et al. supra). The notochord isknown as a source of inductive signals that contribute to theregionalization of the neural plate (Yamada et al., (1991) Cell64:635-647); Ericson et al., (1992) Science 256:1555-1560). A putativerole of the notochord in the early inductive events leading to theregionalization of the gut endoderm can now be studied using Ipf1 as amarker.

[0148] Since the onset of Ipf1 expression (e8.5) is correlated with thecommitment of the gut endoderm to a pancreatic fate, this early patternof Ipf1 expression may reflect the specification of pluripotentpancreatic stem cells that are the progenitors of all the variouspancreatic cells. However, it has recently been shown that hormone genetranscripts are present at 20 somites, prior to morphogenesis, and thatexocrine gene expression is initiated well after the formation of thepancreatic diverticulum (Gittes et al., supra). These results, whichindicate that the endocrine cells are specified before the exocrineones, may suggest that the early Ipf1-expressing cells are theprogenitors only of the β-cells rather than of all pancreatic cells.

[0149] While it is not clear that there exists a precise lineagerelationship between the different pancreatic hormone-producing cells, anumber of independent studies of normal embryonic islet cells and ofdifferent islet tumor cell lines have shown that certain hormones can beco-expressed in the same cells (Yoshinari et al. (1992) Anat Embryol105:63-70; Madsen et al., (1986) J Cell Biol 103:2025-2034; Teitelman etal., (1987) Dev Biol 121:454-466; Alpert et al., (1988) Cell 53:295-308:Herrera et al., (1991) Development 113:1257. These studies have alsosuggested that the terminal differentiation of individual islet celltypes occurs late in development. There is not complete agreement onwhich of the hormones can be colocalized, but taken together the resultsfavor the hypothesis of a common 1 pancreatic endocrine cell lineage(Alpert et al., supra; Herrera et al., supra; Gittes et al. supra). Ithas been suggested that at e15.5, about half of the insulin-producingcells also express glucagon but a different study claims that in themouse there is never any co-expression of these two hormones. Since, asdescribed below, Ipf1 becomes restricted to the insulin-producing cellsvery early in development and since no apparent co-expression of Ipf1andglucagon is observed, these results also suggest that the α- and β-cellsdevelop independently.

[0150] It is noted that expression of XlHbox8 is also restricted toepithelial cells of the duodenum and the developing pancreas, but inadults XlHbox8 is only found in the nuclei of the pancreatic excretoryducts: no expression is evident in the pancreatic islet cells (Wright etal., (1988) Development 104:787-794. The differences in amino acidsequence and in the pattern of expression of these proteins suggest thateither Ipf1 is a mouse homolog of XlHbox8 which has diverged both withrespect to structure and function, or that there exist at least tworelated homeodomain proteins which are both involved in pancreasdevelopment. By using affinity purified antibodies, the experimentsdescribed below have avoided cross-reaction with a putative XlHbox8mouse homolog. Moreover, Ipf1 genomic DNA has been isolated andcharacterized, but by using the Ipf1 homeobox probe in low stringencyhybridization these experiments so far failed to detect any Ipf1-relatedgene. Similarly, the Xenopz's DNA fragment encoding the C-terminal partof the XlHbox8 gene has been isolated but no cross-hybridization hasbeen detected with the mouse genomic DNA fragment. However, since theseresults are negative, the possibility that the mouse genome contains atrue XlHbox8 homolog cannot be excluded, and likewise, neither canalternative splicing be excluded as a way of generating differentpolypeptides having an identical homeodomain.

[0151] Other homeodomain proteins, like members of the POU, LIM andNkx-2 families, are expressed at high levels in subsets of adult celltypes and are implicated in transcriptional control in terminallydifferentiated cells (Herr et al.,(1988) Genes Dev 2:1515-1516; Freyd etal., (1990) Nature 344:875-878; Karlsson et al., (1990) Nature344:879-882; Price et al., (1992) Neuron 8:241-255). In some aspects,Ipf1 resembles the POU Pit-I/GHF-I protein in that both proteins areselectively expressed in polypeptide hormone-producing cells ndtranscriptionally regulate specific hormone genes (Bodner et al., (1988)Cell 55:505-518; Ingraham et al., (1988) Cell 55:519-529). Mutations ofthe Pit-I/GHF-I gene in dwarf mice result in hypoplasia of thePit-expressing cells, providing evidence for a role of Pit-I inspecification of these cell types (Li et al., (1990) Nature347:528-533). It is proposed that Ipf1 may have a similar function inthe development of the pancreas.

[0152] Moreover, temporal expression pattern of IPF1 resembles that ofthe lymphoid-specific transcriptional factor Ikaros, the RNA for whichis highly expressed in the early fetal liver and which then starts todecline at e14 (Georgopoulos et al., (1992) Science 258:808-812). It hasbeen argued that the early high level expression of Ikaros is necessaryfor further commitment and differentiation of the pluripotenthematopoietic stem cell, and it has been suggested that the decrease inexpression represents changes in the developmental profile ofhematopoietic progenitors towards a more committed erythroid stage(Georgopoulos et al., supra). Ipf1 may have a similar dual function inthe development of the pancreas and the β-cells.

[0153] i) Cloning of cDNAs encoding IPF1

[0154] The islet-cell specific expression of the rat insulin I gene isdependent both on a distal enhancer element and on more proximal“promoter” sequences which do not contribute to the enhancing activity.The rat insulin I gene, for example, contains a short DNA element,TAATGGG, which is located at positions −80 to −74 and which is conservedin the rat, mouse, guinea pig and human insulin promoters (Steiner etal., (1985) Anna Rev Genet).

[0155] To isolate the gene encoding the putative transcriptionalregulatory protein which binds this site, a set of degenerate PCRprimers were designed that were complementary to a consensus sequence ofhelix 3 of known homeodomain proteins (see Materials and methods).Lacking any information on the possible structure of IPF1, a primercomplementary to sequences in the λgt11 vector was used as the secondprimer in the PCR. These two sets of primers were used in PCR on totalphage DNA prepared from a phage stock of a βTC1 λgt11 cDNA library. Bycloning and sequencing the DNA fragments obtained in the PCR, a 100 bpfragment was identified which showed an open reading frame encoding apartial homeodomain. Using this fragment as a probe, overlapping cDNAsencoding a protein of 284 amino acids with a calculated molecular weightof 31 kDa were isolated from the same library.

[0156] The encoded protein IPF1 was so named for reasons presentedbelow. The deduced amino acid sequence revealed a homeodomain which isdivergent from the Antennapedia prototype and which contained a uniquehistidine in position 45 of helix 3 (His-190, SEQ ID No. 2). Thishomeodomain is not identical to any previously isolated mammalianhomeodomain protein, but part of the homeodomain is identical to theknown part of the homeodomain of the XlHbox8 protein from Xenopus laevis(Wright et al., (1988) 104:787-794) (FIG. 1B). Only the C-terminal part,including roughly two-thirds of the homeodomain, of XlHbox8 has beenreported. No homology outside of the homeodomain is observed betweenthese two proteins. A genomic DNA fragment has been isolated from theleech Helobdella triseralis, which encodes a homeodomain sharing somehomology with the IPF1 and XlHbox8 proteins (Weeden et al., (1990) Nu.Acid Res 18:1908). Only the sequence of the homeodomain of this protein,Htr-A2, has been published but it is 86% homologous to td the IPF1homeodomain and has the characteristic histidine in helix 3. Noadditional information is available regarding this protein.

[0157] RNA prepared from the IPF1 cDNA template was translated in vitroand the DNA binding specificity of the in vitro translation product wasdetermined using an electrophoretic mobility shift assay (EMSA) and theinsulin promoter PI site as a probe (Ohisson et al., (1991) Mol Endocrin5:897-904; see also Materials and Methods below). The in vitrotranslation product bound to the P1 element and migrated to the samerelative position in the gel as IPF1 from the βTC1 nuclear extract.Competition studies with wild-type and mutant P1 sites showed that thein vitro translation product had the same binding specificity as theendogenous IPF1.

[0158] As described below, antibodies were raised against the C-terminalhalf of the encoded protein, carrying 48 amino acids of the homeodomain.The obtained antiserum was shown to block binding of nuclear Ipf1 to thePI site, but did not recognize other homeodomain proteins like Isl-1(Karlsson et al.; (1990) Nature 344:879-882). To show that the clonedcDNA encoded IPF1, antibodies directed against the part of Ipf1 locatedC-terminally to the homeodomain were affinity purified using theglutathione S-transferase (GST) gene fusion system (see Materials andmethods). These affinity purified antibodies, which recognize theC-terminal part but not the homeodomain of IPF1. gave rise to asupershifted complex of nuclear IPF1 bound to the PI site. Collectively,these results indicate that the isolated cDNA encodes IPF1.

[0159] ii) Ipf1 Transactivates the Insulin Promoter

[0160] Sequences immediately upstream of the insulin gene TATA box,which include the PI promoter site, have previously been shown to be ofimportance for the transcriptional activity of the insulin 5′ flankingDNA and to be preferentially active in pancreatic endocrine cell lines(Edlund et al., (1985) Science 230:912-916. It is demonstrated hereinthat a 5′ flank where the AA residues in the TAATGGG IPF1 binding sitehave been changed to CC, and, to which IPF1 fails to bind, has a2.5-fold lower. activity than the wild-type 5′ flank in PTC1 cells (FIG.1A). This result is in contrast to previously published results(Karlsson et al., (1987) PNAS 84:8819-8823). The activity of thewild-type insulin 5′ flank in PTC1 cells was farther increased byco-transfection with a vector in which Ipf1 expression is under thecontrol of the Rous sarcoma virus (RSV) long terminal repeat (FIG. 1A)and, as expected, the mutant 5′ flank could not be transactivated byIpf1. Ipf1 was also tested to see if it could transactivate a constructcarrying five copies of the P1 site linked to the β-globin TATA box innon-pancreatic cells. If Ipf1 could transactivate this construct, itshould be preferentially active in Ipf1-containing insulin-producingcells. Therefore, the intrinsic cell specificity of this construct wasanalyzed and found that it was, relative to the control TATA boxconstruct, 3-fold more active in the βTC1 cells than in the CHO cells(FIGS. 1B and 1C). By expressing Ipf1 in the CHO cells, the activity ofthe 5× P1 construct was increased to that seen in the PTC1 cells (FIG.1B). The activity of the 5× P1 construct could also be increased 2-foldin the βTC1 cells by co-transfection with the RSV-Ipf1 expression vector(FIG. 1C). As a specificity control, an RSV-Isl-1 expression constructwas shown to not be able to transactivate the 5× P1 β-globin constructin the CHO cells (FIG. 1B).

[0161] iii) Ipf1 is selectively expressed in the adult pancreaticβ-cells

[0162] Native Ipf1 is detected in nuclear extracts prepared frominsulin-producing OTC 1 cells but not in nuclear extracts prepared fromglucagon-producing αTC1 cells or from non-endocrine cells. UtilizingNorthern analysis of RNA prepared from αTC1 cells, βTC1 cells, and avariety of other mouse cell lines and organs, a 2.3 kb IPF1 transcriptwas detected only in PTC1 cells. Ipf1 RNA was also found to be presentin insulin-producing cell lines from other species. As a test of thedifferentiated state of the αTC1 and βTC1 cells used, RNA from thesecells was probed with insulin and glucagon cDNA.IT was observed thatvery little or no co-expression of these genes occurs.

[0163] The pattern of expression of IPF1 in adult mouse pancreas wasanalyzed at the single cell level by immunohistochemistry using affinitypurified anti-Ipf1 antibodies (see Materials and methods).Immunoreactivity was readily detectable within the islets whereas nostaining was observed in the exocrine pancreas or within the duct cells.Within the islets, the staining paralleled the typical pattern forinsulin-producing cells since the majority of the cells were positiveand were all located in the center of the islets and doubleimmunostaining using anti-IPF1 and anti-hormone antibodies showed thatIpf1 was not present in glucagon and somatostatin-producing cells. SinceIPF1 is apparently restricted to the β-cells of adult pancreas and sinceit binds to and transactivates the insulin promoter, it is very likelythat IPF1 is directly involved in the control of the β-cell specificactivity of the insulin gene.

[0164] iv) Ipf1 is Selectively Expressed in the Pancreatic ProgenitorCells in Early Mouse Embryos

[0165] The affinity purified anti-Ipf1 antibodies were employed forimmunohistochemistry on cryostat sections of mouse e8.5-15.5 embryos tostudy the temporal and spatial pattern of IPF1 expression at the singlecell level. At all stages of development, Ipf1 expression was onlydetected in the pancreatic anlagen or in the pancreas itself. In bothsagittal and transverse sections of 18-20 somite embryos, IPF1 positivecells are present in the part of duodenum which will later give rise tothe dorsal and ventral pancreas. By using whole-mountimmunohistochemistry (see Materials and methods) it was conclusivelydemonstrated that Ipf1 is only expressed in the dorsal and ventral wallsof the duodenum and not in the lateral parts of the gut wall. Thus, Ipf1expression is restricted to the sites where the dorsal and ventralpancreas will start to evaginate. At the 18-20 somite stage the majorityof the cells in these two regions are IPF1 positive. Moreover, a fewIPF1 positive cells can be detected as early as the 13 somite stage(e8.5) in both the dorsal and ventral walls of the duodenum, whereas noIpf1 positive cells were detected at the 10 somite stage.

[0166] In the mouse pancreas a few insulin-containing cells appeararound e12 in the dorsal bud and a day or so later in the ventral bud.Glucagon-containing cells are already present at e10.5 in the dorsal bud(Herrera et al., (1991) Development 113:1257-1265). Using anti-hormoneantisera and affinity purified anti-Ipf1 antibodies, the pattern ofexpression of Ipf1 was correlated with that of glucagon and insulin. Itwas observed that there was a drastic decrease in the relative number ofIpf1-expressing cells between e 10.5 and e 11.5. At el 3.5 there werestill very few Ipf1 expressing cells and none or very few of theglucagon-expressing cells express Ipf1. This relative decrease in thenumber of Ipf1 positive cells is most likely the result of ingrowth ofthe exocrine parenchyma which would result in the dispersion of the Ipf1positive cells. At e15.5, the relative number of Ipf1 positive cells hasincreased substantially and at this stage the pancreas contains bothinsulin- and glucagon-producing cells but apparently only theinsulin-producing cells express Ipf1. The increase in the relativenumber of Ipf1-expressing cells between e13.5 and e15.5 correlates witha previous observation of a 20-fold relative increase in the number ofinsulin-producing β-cells during this period (Herrera et al., supra).

[0167] v) Materials And Methods

[0168] Polymerase Chain Reaction and Isolation of cDNA Clones

[0169] The following combinations of oligonucleotides were used in thePCRs: a set of degenerate oligonucleotides complementary to a consensussequence of helix III of known homeoboxes,5′-GCAAGCTTCATICT/GICT/GG/ATTCITTGG/AAACCA-3′, was combined with eitherof the two oligonucleotides included in the λgt11 insert screeningamplimer set (cat. no 5412-1, Clontech Laboratories Inc., Palo Alto,Calif.). The DNA template was prepared from a αTC1 λgt11 library (Walkeret al., (1990) Nuc. Acid Res. 18:1109-1176. An aliquot of this librarywas dialyzed against distilled water and then frozen, thawed and used inthe PCRs which were carried out using Taq DNA polymerase(Perkin-Elmer/Cetus) according to the manufacturer's instructions. ThePCR product of interest was sequenced and subsequently labelled with[α-³²P]dATP and used as a probe to screen the βTC )λgt11 in order toisolate a full-length cDNA clone.

[0170] Nuclear Extract Preparation and DNA Transfections

[0171] Nuclear extract was prepared from OTC1 cells, a transgenicallyderived insulin-producing β-cell line (Efrat et al., (1988) PNAS85:9037-9041), as previously described (Ohlsson and Edlund, (1986) Cell45:3544). DNA transfections of βTC1 and CHO cells were carried out asdescribed previously (Walker et al., (1983) Nature 306:557-581).

[0172] In vitro Transcription and Translation

[0173] The Isl-1 template for SP6 polymerase-directed in vitrotranscription has been described earlier (Ohlsson et al., (1991) MolEndocrinol 5:897-904). The IPF1 template was constructed by insertingthe full-length Ipf1 cDNA into the vector pGEM 3. The template waslinearized before T7 polymerase-directed in vitro transcription. Invitro translation in rabbit reticulocyte lysates was carried out asrecommended by the manufacturer (Promega, Madison, Wis.).

[0174] Electrophoretic Mobility Shift Assay

[0175] The following oligonucleotides were used in the EMSA: wild-typepromoter element P1: GCCCTTAATGGGCCAAACGGCA; P1 mutant 1:GGGGTTAATGGGCCAAACGGCA; P1 mutant 2: GCCCTTCCTGGGCCAAACGGCA; P1 mutant3: GCCCTTAATCCCCCAAACGGCA; and wild-type enhancer element E2:GCCCCTTGTTAATAATCTAAT (Ohlsson et al., (1991), supra). Theseoligonucleotides were all custom-made by Symbicom AB (Umea, Sweden). Theoligonucleotides were annealed, end-labelled and purified as previouslydescribed (Ohlsson et al., (1988), supra). The EMSA was carried out asdescribed previously (Ohlsson et al., (1988), supra). The antisera usedwere added together with nonspecific DNA {poly[d(I-C)]:poly[d(A-T)], 1:1ratio} 15-20 min before the specific end labelled synthetic DNAfragment.

[0176] Northern blot analysis

[0177] Poly(A)+ RNA was prepared from the following cell lines: βTC1,αTC1 [a transgenically derived glucagon-producing α-cell line (Efrat etal. (1988) Neuron 1:605-613)], Ltk- (a mouse fibroblast cell line) andJ558L [a mouse myeloma (Oi et al., (1983) PNAS 80:825-829)] using theFast Track kit from Invitrogen Inc. (San Diego, Calif.). Poly(A)+ RNAsfrom the tissues used were purchased from Clontech Inc. (Palo Alto,Calif.). Electrophoresis of RNA, blotting, stripping, hybridization andrandom labelling of probes were performed as described previously(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, supra).

[0178] Preparation of Antisera

[0179] Anti-IPF1 antiserum was prepared using a DNA fragment encodingthe C-terminal half of IPF1 which includes part of the homeodomain. Thisfragment was inserted into the expression vector path 11 and expressedas a TrpE fusion protein (Klempnauer and Sippel, (1987) EMBO J6:2719-2725; Angel et al., (1988) Nature 332:166-171). The fusionprotein was purified by preparative SDS-PAGE and used to elicitpolyclonal antibodies in rabbits (Thor et al., (1991) Neuron 7:1-9). Toobtain antibodies that specifically recognized the C-terminal part ofIpf1, the C-terminal part of Ipf1 (amino acids 215-284) lacking anyhomeodomain residues was expressed as a fusion protein with glutathioneS-transferase using the GST gene fusion system in Escherchia coli (Smithand Johnson, (1988) Gene 67:31-40; Pharmacia, Uppsala, Sweden). Thefusion protein was affinity purified on a glutathione-Sepharose 4Bcolumn (Pharmacia, Uppsala, Sweden) and the eluted fusion protein wasimmobilized on Affi-gel 10 (Thor et al., supra). The anti-Ipf1 antiserumwas applied to a column containing the immobilized Ipf1 C-terminalfusion protein; after extensive washing, the bound antibodies wereeluted, reapplied to an identical column and subsequently eluted (Thoret al., supra).

[0180] Immunohistochemistry

[0181] Immunohistochemistry on adult mouse pancreas was done on freshlyfrozen mouse C57BL/6JBom (Bomholtgard Breeding and Research Centre Ltd,Ry, Denmark) pancreas that had been sectioned on a cryostat.Cryosections (8 μm) were mounted on glass slides. air dried and storedat −80° C. Prior to immunostaining, the sections were fixed in 1%paraformaldehyde (pH 7.4) for 20 min, washed in TBS (50 mM Tris-HCl pH7.4, 150 mM NaCl) and blocked with 5% normal goat serum in TBST (TBScontaining 0.1% Triton X-100) for 10 min. Sectioning of embryos was doneby harvesting embryos from timed (Kaufman, 1992), pregnant C57BL/6JBornmice that were either fixed in 1% paraformaldehyde (pH 7.4) for 1-2 hand then frozen (for e8.5-11.5 embryos) or frozen directly (fore12.5-16.5 embryos). The immunohistochemistry on both pancreas andembryos was then carried out as previously described (Thor et al.,supra).

[0182] Whole-mount Immunohistochemistry

[0183] Whole-mount immunohistochemistry was carried out on e8.5-9.5mouse embryos as described previously (Ruiz I Altaba and Jessel, (1991)Development 1 12:945-958) but with the following modifications. Embryoswere fixed in 1% paraformaldehyde, 0.1 M potassium phosphate pH 7.4 for1-2 h, transferred to 30% sucrose, 0.1 M potassium phosphate, 0.02%sodium azide and stored at +4 C. Before staining, the embryos weretransferred to TBS for 1 h. The embryos were then blocked for endogenousperoxidase activity in methanol containing 3% hydrogen peroxide for atleast 2 h. The blocking solution was then gradually replaced by TBS.Non-specific binding was reduced by incubation in 5% normal goat serumin TBST. Antibodies were diluted in TBST with 5% normal goat serum. Theprimary antibodies were detected with the ABC immunoperoxidase systemaccording to the manufacturer's recommendation (Vector LaboratoriesInc., USA) with the exception that the ABC complex was diluted 5-foldbefore incubation. After each antibody incubation, embryos wereextensively washed in TBST for at least 2 h with four to six changes.

EXAMPLE 2 Ipf1 Transgenic Mice

[0184] In mouse embryos, Ipf1-expression is restricted to the developingpancreatic anlagen and is initiated when the foregut endoderm commits toa pancreatic fate. It is now demonstrated that mice homozygous for atargeted mutation in the Ipf1 gene selectively lack a pancreas. Themutant pups survive fetal development but die within a few days afterbirth. The gastrointestinal part and all other internal organs werenormal in appearance. No pancreatic tissue and no ectopic expression ofinsulin or pancreatic amylase could be detected in mutant embryos andneonates. These findings show that Ipf1 is needed for the formation ofthe pancreas and suggest that Ipf1 acts to determine the fate of commonpancreatic precursor cells and/or to regulate their propagation.

[0185] The mammalian pancreas is a mixed exocrine and endocrine glandthat, in most species, arises from ventral and dorsal buds whichsubsequently merge to form the definitive pancreas. In both mouse andrat, the first histological sign of morphogenesis of the dorsal pancreasis a dorsal evagination of the duodenum at the level of the liver ataround 22-25 somite stage, and shortly thereafter a ventral evaginationappears as a derivative of the liver diverticulum 2-4. Low levels ofinsulin gene transcripts are already present and restricted to thedorsal foregut endoderm at 20 somites suggesting that pancreas orinsulin-gene-specific transcriptional factors are present in this regionprior to the onset of morphogenesis.5

[0186] In early mouse embryos, the Ipf1 protein is detected only in thedeveloping pancreas but alter in development and in adult mouse pancreasIpf1 is selectively expressed in the (3-cells where it binds to andtransactivates the insulin gene. The structurally related XenopusXIHbox86 and rat STF-1/IDX-17,8 proteins, are also selectively expressedin the endoderm of the duodenum and the pancreas but at present it isnot known if these proteins represent functional homologs of Ipf1. Totest the hypothesis that Ipf1 plays a role in the pancreatic commitmentof the foregut endoderm, Ipf1-deficient mice were generated by deletingexon 2, which encodes the homeodomain of Ipf1 using homologousrecombination in ES-cells (FIG. 2). Mice heterozygous for the Ipf1mutation show no apparent abnormalities, they are fertile and theiroffspring show the expected Mendelian frequencies of mutant genotypesindicating that the Ipf1-deficiency does not cause embryonic lethality.However, all homozygous mutant mice die within a few days after birth,showing a complete penetrance of this neonatal mortality phenotype. Thetargeted Ipf1-/- mutant embryos show no detectable Ipf1 immunoreactivityas analyzed by whole-mount immunohistochemistry using anti-Ipf1antibodies.

[0187] Newborn homozygous mutant mice do not show any morphologicalabnormalities, except that they appear slightly smaller than wildtypeand heterozygous littermates, on average 80% for newborn pups (n=15),and 60% for two day old pups (n=15). Most Ipf1-deficient pups areinitially able to feed as indicated by the presence of milk in theirstomachs, but all die within a few days after birth. To determine ifpancreas development was affected in the Ipf1 mutants, histologicalanalyses were performed on new born pups from a cross betweenheterozygous Ipf1 mutants. The homozygous Ipf1 mutants completely lack apancreas but the duodenum from which the pancreas normally developsshowed the normal C-shaped form. The intestines of the Ipf1-/- pups(n=8) have the same relative length (cm/g body weight) +/−10%, as thewildtype pups and show no apparent abnormalities except that the loopsof the small intestine are positioned somewhat differently in theabdomen compared to the wildtype. In the homozygote mutants (n=8) boththe liver. which develops from the same part of the primitive foregut asthe pancreas, and the spleen, which is thought to be derived from“pancreatic” mesoderm, also appear normal and show the same relativeweight (mg/g body weight) +/−10%, as the wildtype. The common bile ductand the ventral pancreatic duct are both derived from the hepaticdiverticulum of the foregut and the main duct of the pancreas normallyfuses with the common bile duct in the duodenal wall and both empty intothe duodenal lumen at the major duodenal papilla. In the homozygousmutants there is no pancreatic duct, but the common bile duct ispresent, indicating that, apart from the lack of a pancreas, theduodenal tract is normally developed. Thus, it may be concluded thatIpf1-deficiency leads to the selective loss of the pancreas. The Ipf1-/-pups that are able to feed and live for more than 2 days show elevatedurine glucose levels, >55 mM for three day old Ipft-/- pups (n=7),suggesting that the cause of death is partly due to insulin deficiency.The lack of the other islet hormones and the exocrine digestive enzymesmay also contribute to the pathology.

[0188] The complete lack of a pancreas indicates that Ipf1 is requiredearly in the development of the pancreas and suggests that Ipf1 actseither at the level of determination or the early differentiation of thepancreas. In normal mice, pancreatic amylase and insulin are highly andspecifically expressed in the exocrine and endocrine pancreas,respectively, and the expression of the gut-hormone gastrin can be usedto determine the state of differentiation of the intestinal epithelium.To exclude the possibility that in the Ipf1-deficient mice morphogenesiswas arrested but cytodifferentiation still occurred, immunohistochemicalanalysis of mutant and wild-type mouse embryos and neonates wasperformed. In the mouse, both insulin and amylase expressing cells haveaccumulated in sufficiently high numbers in the pancreas at aroundembryonic day e15 to allow reproducible detection byimmunohistochemistry. The intestinal epithelium differentiates late indevelopment so expression of gastrin was monitored, in sections of theduodenum, in newborn animals. No pancreatic tissue was present in mutante15 embryos and neonates and no ectopic expression of insulin andamylase was detected in serial sagital sections of the duodenum ofmutant embryos and neonates. Cells expressing gastrin were present inthe duodenum from both wildtype and mutant newborn animals. This, andthe normal histology of the intestinal epithelium in the mutantsindicate that this part of the duodenum develops normally. The lack ofpancreatic tissue and of ectopic expression of insulin and pancreaticamylase in the developing duodenum show that both cytodifferentiationand morphogenesis of the pancreas is arrested in the homozygous mutants.

[0189] The observed phenotype further suggests that Ipf1 has an earlyfunction in the initial stages of pancreas development. A few Ipf1positive cells can first be detected in the gut region at around the10-12 somite stages which is when the foregut endoderm commits to apancreatic fate. This and the lack of a pancreas in the Ipf1-deficientmutants strongly suggest that Ipf1 functions in the determination and/ormaintenance of the pancreatic identity of common precursor cells, or inthe regulation of their propagation.

[0190] All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

[0191] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 9 1313 base pairs nucleic acid both linear cDNA CDS 128..979 1CAGGAGAGCA GTGGAGAACT GTCAAAGCGA TCTGGGGTGG CGTAGAGAGT CCGCGAGCCA 60CCCAGCGCCT AAGGCCTGGC TTGTAGCTCC GACCCGGGGC TGCTGGCCCC AAGTGCCGGC 120TGCCACC ATG AAC AGT GAG GAG CAG TAC TAC GCG GCC ACA CAG CTC TAC 169 MetAsn Ser Glu Glu Gln Tyr Tyr Ala Ala Thr Gln Leu Tyr 1 5 10 AAG GAC CCGTGC GCA TTC CAG AGG GGC CCG GTG CCA GAG TTC AGC GCT 217 Lys Asp Pro CysAla Phe Gln Arg Gly Pro Val Pro Glu Phe Ser Ala 15 20 25 30 AAC CCC CCTGCG TGC CTG TAC ATG GGC CGC CAG CCC CCA CCT CCG CCG 265 Asn Pro Pro AlaCys Leu Tyr Met Gly Arg Gln Pro Pro Pro Pro Pro 35 40 45 CCA CCC CAG TTTACA AGC TCG CTG GGA TCA CTG GAG CAG GGA AGT CCT 313 Pro Pro Gln Phe ThrSer Ser Leu Gly Ser Leu Glu Gln Gly Ser Pro 50 55 60 CCG GAC ATC TCC CCATAC GAA GTG CCC CCG CTC GCC TCC GAC GAC CCG 361 Pro Asp Ile Ser Pro TyrGlu Val Pro Pro Leu Ala Ser Asp Asp Pro 65 70 75 GCT GGC GCT CAC CTC CACCAC CAC CTT CCA GCT CAG CTC GGG CTC GCC 409 Ala Gly Ala His Leu His HisHis Leu Pro Ala Gln Leu Gly Leu Ala 80 85 90 CAT CCA CCT CCC GGA CCT TTCCCG AAT GGA ACC GAG CCT GGG GGC CTG 457 His Pro Pro Pro Gly Pro Phe ProAsn Gly Thr Glu Pro Gly Gly Leu 95 100 105 110 GAA GAG CCC AAC CGC GTCCAG CTC CCT TTC CCG TGG ATG AAA TCC ACC 505 Glu Glu Pro Asn Arg Val GlnLeu Pro Phe Pro Trp Met Lys Ser Thr 115 120 125 AAA GCT CAC GCG TGG AAAGGC CAG TGG GCA GGA GGT GCT TAC ACA GCG 553 Lys Ala His Ala Trp Lys GlyGln Trp Ala Gly Gly Ala Tyr Thr Ala 130 135 140 GAA CCC GAG GAA AAC AAGAGG ACC CGT ACT GCC TAC ACC CGG GCG CAG 601 Glu Pro Glu Glu Asn Lys ArgThr Arg Thr Ala Tyr Thr Arg Ala Gln 145 150 155 CTG CTG GAG CTG GAG AAGGAA TTC TTA TTT AAC AAA TAC ATC TCC CGG 649 Leu Leu Glu Leu Glu Lys GluPhe Leu Phe Asn Lys Tyr Ile Ser Arg 160 165 170 CCC CGC CGG GTG GAG CTGGCA GTG ATG TTG AAC TTG ACC GAG AGA CAC 697 Pro Arg Arg Val Glu Leu AlaVal Met Leu Asn Leu Thr Glu Arg His 175 180 185 190 ATC AAA ATC TGG TTCCAA AAC CGT CGC ATG AAG TGG AAA AAA GAG GAA 745 Ile Lys Ile Trp Phe GlnAsn Arg Arg Met Lys Trp Lys Lys Glu Glu 195 200 205 GAT AAG AAA CGT AGTAGC GGG ACC CCG AGT GGG GGC GGT GGG GGC GAA 793 Asp Lys Lys Arg Ser SerGly Thr Pro Ser Gly Gly Gly Gly Gly Glu 210 215 220 GAG CCG GAG CAA GATTGT GCG GTG ACC TCG GGC GAG GAG CTG CTG GCA 841 Glu Pro Glu Gln Asp CysAla Val Thr Ser Gly Glu Glu Leu Leu Ala 225 230 235 GTG CCA CCG CTG CCACCT CCC GGA GGT GCC GTG CCC CCA GGC GTC CCA 889 Val Pro Pro Leu Pro ProPro Gly Gly Ala Val Pro Pro Gly Val Pro 240 245 250 GCT GCA GTC CGG GAGGGC CTA CTG CCT TCG GGC CTT AGC GTG TCG CCA 937 Ala Ala Val Arg Glu GlyLeu Leu Pro Ser Gly Leu Ser Val Ser Pro 255 260 265 270 CAG CCC TCC AGCATC GCG CCA CTG CGA CCG CAG GAA CCC CGG 979 Gln Pro Ser Ser Ile Ala ProLeu Arg Pro Gln Glu Pro Arg 275 280 TGAGGACAGC AGTCTGAGGG TGAGCGGGTCTGGGACCCAG AGTGTGGACG TGGGAGCGGG 1039 CAGCTGGATA AGGGAACTTA ACCTAGGCGTCGCACAAGAA GAAAATTCTT GAGGGCACGA 1099 GAGCCAGTTG GATAGCCGGA GAGATGCTGCGAGCTTCTGA AAAAACAGCC CTGAGCTTCT 1159 GAAAACTTTG AGGCTCGCTC TGATGCCAAGCTAATGGCCA GATCTGCCTC TGAGGACTCT 1219 TTCCTGGGAC CAATTTAGAC AACCTGGGCTCCAAACTGAG GACAATAAAA AGGGTACAAA 1279 CTTGAGCGTT CCAATACGGA CCAGCAGGCGAGAG 1313 284 amino acids amino acid linear protein 2 Met Asn Ser GluGlu Gln Tyr Tyr Ala Ala Thr Gln Leu Tyr Lys Asn 1 5 10 15 Pro Cys AlaPhe Gln Arg Gly Pro Val Pro Glu Phe Ser Ala Asn Pro 20 25 30 Pro Ala CysLeu Tyr Met Gly Arg Gln Pro Pro Pro Pro Pro Pro Pro 35 40 45 Gln Phe ThrSer Ser Leu Gly Ser Leu Glu Gln Gly Ser Pro Pro Asp 50 55 60 Ile Ser ProTyr Glu Val Pro Pro Leu Ala Ser Asp Asp Pro Ala Gly 65 70 75 80 Ala HisLeu His His His Leu Pro Ala Gln Leu Gly Leu Ala His Pro 85 90 95 Pro ProGly Pro Phe Pro Asn Gly Thr Glu Pro Gly Gly Leu Glu Glu 100 105 110 ProAsn Arg Val Gln Leu Pro Phe Pro Trp Met Lys Ser Thr Lys Ala 115 120 125His Ala Trp Lys Gly Gln Trp Ala Gly Gly Ala Tyr Thr Ala Glu Pro 130 135140 Glu Glu Asn Lys Arg Thr Arg Thr Ala Tyr Thr Arg Ala Gln Ser Ser 145150 155 160 Glu Leu Glu Lys Glu Phe Leu Phe Asn Lys Tyr Ile Ser Arg ProArg 165 170 175 Arg Val Glu Leu Ala Val Met Leu Asn Leu Thr Glu Arg HisIle Lys 180 185 190 Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys GluGlu Asp Lys 195 200 205 Lys Arg Ser Ser Gly Thr Pro Ser Gly Gly Gly GlyGly Glu Glu Pro 210 215 220 Glu Gln Asp Cys Ala Val Thr Ser Gly Glu GluLeu Leu Ala Val Pro 225 230 235 240 Pro Leu Pro Pro Pro Gly Gly Ala ValPro Pro Gly Val Pro Ala Ala 245 250 255 Val Arg Glu Gly Leu Leu Pro SerGly Leu Ser Val Ser Pro Gln Pro 260 265 270 Ser Ser Ile Ala Pro Leu ArgPro Gln Glu Pro Arg 275 280 29 base pairs nucleic acid single linearcDNA 3 GCAAGCTTCA TNCKNCKRTT YTGRAACCA 29 22 base pairs nucleic acidsingle linear cDNA 4 GCCCTTAATG GGCCAAACGG CA 22 22 base pairs nucleicacid single linear cDNA 5 GGGGTTAATG GGCCAAACGG CA 22 22 base pairsnucleic acid single linear cDNA 6 GCCCTTCCTG GGCCAAACGG CA 22 22 basepairs nucleic acid single linear cDNA 7 GCCCTTAATC CCCCAAACGG CA 22 21base pairs nucleic acid single linear cDNA 8 GCCCCTTGTT AATAATCTAA T 2116 base pairs nucleic acid single linear cDNA 9 GCCCTTAATG GGCCAA 16

I claim:
 1. A substantially pure preparation of an Ipf1 polypeptide having a sequence identical or homologous to an amino acid sequence represented in SEQ ID No.
 2. 2. The Ipf1 polypeptide of claim 1, which polypeptide modulates at least one of proliferation, differentiation or survival of a cell which expresses a gene that is transcriptionally regulated by an Ipf1-responsive element (Ipf1-RE).
 3. The Ipf1 polypeptide of claim 2, wherein the Ipf1-RE comprises a P1 promoter having a nucleotide sequence TAATGGG.
 4. The Ipf1 polypeptide of claim 2, wherein the cell is a pancreatic cell.
 5. The Ipf1 polypeptide of claim 4, wherein the pancreatic cell is a K-islet cell.
 6. The Ipf1 polypeptide of claim 2, which polypeptide stimulates expression of the Ipf1-responsive gene.
 7. The Ipf1 polypeptide of claim 2, which polypeptide antagonizes expression of the Ipf1-responsive gene.
 8. The Ipf1 polypeptide of claim 7 which polypeptide either (i) retains DNA binding ability and lacks ability to assemble transcriptionally-competent protein complexes, or (ii) lacks DNA binding ability yet retains the ability to compete with a wild-type form of Ipf1 in binding transcription regulatory proteins.
 9. The Ipf1 polypeptide of claim 1, which polypeptide includes at least 4 amino acid residues from Met-1 through Glu-145 or Ser-212 through Arg-284 of the amino acid sequence represented by SEQ ID No. 2, or a sequence homologous thereto.
 10. An immunogen comprising the Ipf1 polypeptide of claim
 1. in an immunogenic preparation, said immunogen being capable of eliciting an immune response specific for said Ipf1 polypeptide.
 11. An antibody preparation specifically reactive with an epitope of the Ipf1 polypeptide of claim
 1. 12. A recombinant polypeptide comprising an Ipf1 polypeptide sequence identical or homologous with an amino acid sequence represented by SEQ ID No.
 2. 13. The polypeptide of claim 12, which protein is a fusion protein further comprising, in addition to said Ipf1 polypeptide sequence, a second polypeptide sequence having an amino acid sequence unrelated to the amino acid sequence represented by SEQ ID No.
 2. 14. The polypeptide of claim 11, wherein said fusion protein includes, as a second polypeptide sequence, a polypeptide which functions as a detectable label for detecting the presence of said fusion protein or as a matrix-binding domain for immobilizing said fusion protein.
 15. The polypeptide of claim 11, wherein said fusion protein is functional in a two-hybrid assay.
 16. An isolated or recombinant Ipf1 polypeptide having an amino acid sequence crossreactive with an antibody which specifically binds the Ipf1 protein represented by SEQ ID No.2.
 17. An isolated or recombinant Ipf1 polypeptide encoded by a nucleic acid which hybridizes under stringent conditions to a nucleic acid sequence represented in SEQ ID No.
 1. 18. A substantially pure nucleic acid encoding a recombinant polypeptide comprising an Ipf1 polypeptide sequence homologous to an amino acid sequence represented in SEQ ID No.
 2. 19. The nucleic acid of claim 18, wherein said polypeptide modulates at least one of proliferation, differentiation or survival of a cell which expresses a gene that is transcriptionally regulated by an Ipf1-responsive element (Ipf1-RE).
 20. The nucleic acid of claim 18, wherein said nucleotide sequence hybridizes under stringent conditions to a nucleic acid probe having a sequence represented by at least 12 consecutive nucleotides of SEQ ID No.
 1. 21. The nucleic acid of claim 18, wherein said nucleotide sequence hybridizes under stringent conditions to a nucleic acid probe having a sequence represented by at least 12 consecutive nucleotides between nucleotide residues 31 to 562 or 761 to 979 of SEQ ID No.
 1. 22. The nucleic acid of claim 18, further comprising a transcriptional regulatory sequence operably linked to said nucleotide sequence so as to render said nucleic acid suitable for use as an expression vector.
 23. An expression vector, capable of replicating in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of claim
 18. 24. A host cell transfected with the expression vector of claim 23 and expressing said recombinant polypeptide.
 25. A method of producing a recombinant Ipf1 polypeptide comprising culturing the cell of claim 24 in a cell culture medium to express said Ipf1 polypeptide and isolating said Ipf1 polypeptide from said cell culture.
 26. A recombinant transfection system, comprising (i) a gene construct including the nucleic acid of claim 18 and operably linked to a transcriptional regulatory sequence for causing expression of said Ipf1polypeptide in eukaryotic cells, and (ii) a gene delivery composition for delivering said gene construct to a cell and causing the cell to be transfected with said gene construct.
 27. The recombinant transfection system of claim 26, wherein the gene delivery composition is selected from a group consisting of a recombinant viral particle, a liposome, and a poly-cationic nucleic acid binding agent.
 28. A transgenic animal having cells which harbor a transgene comprising the nucleic acid of claim
 18. 29. A transgenic animal in which Ipf1-mediated gene expression is inhibited in one or more tissue of said transgenic animal by one of either expression of an antagonistic Ipf1 polypeptide or disruption of endogenous expression of an Ipf1 gene. 30 The transgenic animal of claim 29, wherein the inhibition of Ipf1-mediated gene expression is constitutive.
 31. The transgenic animal of claim 29, wherein the inhibition of Ipf1-mediated gene expression is conditional.
 32. The transgenic animal of claim 31, wherein the conditional inhibition of Ipf1-mediated gene expression is the result of recombinase-mediated expression of an antagonistic Ipf1 polypeptide or recombinase-mediated disruption of endogenous expression of an Ipf1 gene.
 33. The transgenic animal of claim 31, wherein the expression of an antagonistic Ipf1polypeptide is mediated by a prokaryotic transcriptional regulatory protein.
 34. A recombinant gene comprising a nucleotide sequence homologous with SEQ ID No. 1, or a fragment thereof, said nucleotide sequence operably linked to a transcriptional regulatory sequence in an open reading frame and translatable to a polypeptide capable of modulating at least one of proliferation, differentiation or survival of a cell which expresses a gene that is transcriptionally regulated by an Ipf1-responsive element (Ipf1-RE).
 35. A probe/primer comprising a substantially purified oligonucleotide, said oligonucleotide containing a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of SEQ ID No. 1, or naturally occuring mutants thereof.
 36. The probe/primer of claim 35, which probe hybridizes to at least 10 consecutive nucleotides between nucleotide residues 31 to 562 or 761 to 979 of SEQ ID No.
 1. 37. The probe/primer of claim 35, which probe/primer further comprises a label group attached thereto and able to be detected.
 39. A test kit for detecting cells which contain an Ipf1 mRNA transcript, comprising a probe/primer of claim 35 for detecting, in a sample of cells. a level of nucleic acid encoding an Ipf1 protein.
 40. A method for modulating, in an animal, cell growth, differentiation or survival, comprising administering a therapeutically effective amount of an Ipf1 polypeptide which modulates expression of a gene containing an Ipf1-responsive element (Ipf1-RE).
 41. The Ipf1 polypeptide of claim 40, which polypeptide stimulates expression of the Ipf1-responsive gene.
 42. The Ipf1 polypeptide of claim 40, which polypeptide antagonizes expression of the Ipf1-responsive gene.
 43. The Ipf1 polypeptide of claim 40, which polypeptide is expressed from a gene therapy construct.
 44. The Ipf1 polypeptide of claim 40, which polypeptide is a peptidomimetic of a portion of the polypeptide represented in SEQ ID No.
 2. 45. A method for screening for test compounds that modulate the interaction of an Ipf1polypeptide with other transcriptional regulatory proteins, comprising: i. combining an Ipf1 polypeptide, the transcriptional regulatory protein which binds to Ipf1, and a test compound; and ii. detecting the formation of a complex comprising said Ipf1 polypeptide and said transcriptional regulatory protein, wherein a change in the formation of said complex in the presence of said test compound is indicative of a modulator of the interaction between Ipf1 and a transcriptional regulatory protein.
 46. An assay for screening test compounds that modulate the binding of an Ipf1 polypeptide with an Ipf1-responsive element comprising: i. combining a recombinant Ipf1 polypeptide, a nucleic acid including an Ipf1-responsive element, and a test compound; and ii. detecting the formation of a complex comprising the recombinant Ipf1 polypeptide and said nucleic acid, wherein a change in the formation of said complex in the presence of said test compound is indicative of a modulator of the interaction between Ipf1 and an Ipf1-responsive element.
 47. The assay of claim 46, wherein the recombinant Ipf1 polypeptide is expressed in a cell which harbors a reporter gene, the expression of which is regulated by the Ipf1-responsive element, the interaction of said recombinant Ipf1 and Ipf1-responsive element being detected by observing expression of the reporter gene.
 48. A peptidomimetic of an Ipf1 protein of claim 12 which specifically binds an Ipf1-responsive element.
 49. A peptidomimetic of a portion of an Ipf1 protein of claim 12 which specifically binds a transcriptional regulatory protein.
 50. A method of determining if a subject is at risk for a disorder characterized by abherent cell proliferation or differentiation or expression of a peptide hormone, comprising detecting, in a tissue of said subject, the presence or absence of a genetic lesion characterized by at least one of a mutation of a gene encoding a protein represented by SEQ ID No. 2, or a homolog thereof; and the mis-expression of said gene.
 51. The method of claim 50, wherein detecting said genetic lesion comprises ascertaining the existence of at least one of i. a deletion of one or more nucleotides from said gene, ii. an addition of one or more nucleotides to said gene, iii. an substitution of one or more nucleotides of said gene, iv. a gross chromosomal rearrangement of said gene. v. a gross alteration in the level of a messanger RNA transcript of said gene, vi. the presence of a non-wild type splicing pattern of a messanger RNA transcript of said gene, and vii. a non-wild type level of said protein.
 52. The method of claim 50, wherein detecting said genetic lesion comprises i. providing a probe/primer comprising an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of SEQ ID No. 1 or naturally occuring mutants thereof or 5′ or 3′ flanking sequences naturally associated with said gene; ii. exposing said probe/primer to nucleic acid of said tissue; and iii. detecting, by hybridization of said probe/primer to said nucleic acid, the presence or absence of said genetic lesion.
 53. The method of claim 50, wherein detecting said lesion comprises utilizing said probe/primer to determine the nucleotide sequence of said gene and, optionally, of said flanking nucleic acid sequences.
 52. The method of claim 50, wherein detecting said lesion comprises utilizing said probe/primer to in a polymerase chain reaction (PCR).
 53. The method of claim 50, wherein detecting said lesion comprises utilizing said probe/primer in a ligation chain reaction (LCR).
 54. The method of claim 51, wherein the level of said protein is detected in an immunoassay. 